C-A OPERATIONS PROCEDURES MANUAL

ATTACHMENT

9.2.1.g NSRL Users Experimental Safety Approval Form

  ------------------------- --------------------- ------------------------
  C-A OPM Procedures in                           
  which this Attachment is                        
  used.                                           

  9.2.1                                           

                                                  

                                                  
  ------------------------- --------------------- ------------------------

[Hand Processed Changes]{.underline}

  -------------------------- -------------------------- -------------------------- --------------------------
  [HPC No.]{.underline}      [Date]{.underline}         [Page Nos.]{.underline}    [Initials]{.underline}

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  \_\_\_\_\_\_\_\_\_\_\_\_   \_\_\_\_\_\_\_\_\_\_\_\_   \_\_\_\_\_\_\_\_\_\_\_\_   \_\_\_\_\_\_\_\_\_\_\_\_

  \_\_\_\_\_\_\_\_\_\_\_\_   \_\_\_\_\_\_\_\_\_\_\_\_   \_\_\_\_\_\_\_\_\_\_\_\_   \_\_\_\_\_\_\_\_\_\_\_\_

                                                                                   
  -------------------------- -------------------------- -------------------------- --------------------------

Approved:
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
\_\_\_\_\_

Collider-Accelerator Department Chairman Date

P. Cirnigliaro, A. Emrick

  -----------------------------------------------------------------------
  **[NSRL USERS EXPERIMENTAL SAFETY APPROVAL FORM]{.underline}**

  -----------------------------------------------------------------------

*You must complete this form for all work listed on the proposal, not
for any individual runs*

***.***

***4. EXPERIMENTERS** who will be working on this project. Indicate
Principal Investigator. All experimenters must satisfy all training
requirements for the experiment.*

  -------------- -------------- ------------------ ------------------ -----------------------------------------------------------------------------
  **First Name** **Last Name**  **Affiliation**    **Phone**          **Email**

  **Fiorenza**   **Ianzini**    **University of    **319-335-6637**   [**fiorenza-ianzini@uiowa.edu**](mailto:fiorenza-ianzini@uiowa.edu)
                                Iowa Department of                    
                                Pathology 145B MRC                    
                                -- Iowa City, IA                      
                                52242**                               

  **Elizabeth    **Kosmacek**   **University of    **319-335-6544**   [**ekosmace@engineering.uiowa.edu**](mailto:ekosmace@engineering.uiowa.edu)
  A.**                          Iowa Department of                    
                                BME B180ML - Iowa                     
                                City, IA 52242**                      

  **Christina    **Sjogren**    **University of    **319-335-6544**   [**christina-sjogren@uiowa.edu**](mailto:christina-sjogren@uiowa.edu)
  L.**                          Iowa Department of                    
                                Pathology 145 MRC                     
                                -- Iowa City, IA                      
                                52242**                               
  -------------- -------------- ------------------ ------------------ -----------------------------------------------------------------------------

***5. TASK AND HAZARD ANALYSIS:** **Discuss what you will bring to BNL,
how it will be shipped here, what special precautions you will take when
using it and transporting it on-site, and how it will be disposed of
and/or shipped back to your home institution. Include Radioactive items,
materials and samples, hazardous materials, as well as non-radioactive
and non-hazardous items and materials.***

+-----------------------------------------------------------------------+
| **All materials will be shipped to BNL and back to Iowa City by       |
| carrier**                                                             |
|                                                                       |
| **We will bring:**                                                    |
|                                                                       |
| **Plastic ware**                                                      |
|                                                                       |
| **Cells**                                                             |
|                                                                       |
| **Complete media**                                                    |
|                                                                       |
| **Chemicals to prepare samples for end-points**                       |
|                                                                       |
| **Microscope slides**                                                 |
|                                                                       |
| **Samples will be collected for:**                                    |
|                                                                       |
| **Flow Cytometry (FCM)**                                              |
|                                                                       |
| **Western Blotting**                                                  |
|                                                                       |
| **RT-PCR**                                                            |
|                                                                       |
| **Cellular preparation (microscopic slides)**                         |
|                                                                       |
| **Mutation assay**                                                    |
|                                                                       |
| **Live cell imaging**                                                 |
|                                                                       |
| **Samples will be collected** **according to the following            |
| protocols:**                                                          |
|                                                                       |
| **[COMPLETE MEDIA and BUFFERS]{.underline}**                          |
|                                                                       |
| > **Specific medium for each cell line**                              |
| >                                                                     |
| > **Penicillin/Streptomycin**                                         |
| >                                                                     |
| > **Serum**                                                           |
| >                                                                     |
| > **PBS**                                                             |
| >                                                                     |
| > **EDTA**                                                            |
|                                                                       |
| **[CELL PREPARATION (c/p)]{.underline}**                              |
|                                                                       |
| > ***[Sample collection:]{.underline}***                              |
| >                                                                     |
| > \- resuspend cells in monolayer collecting all the suspension       |
| > fluids (media, PBS and cells                                        |
| >                                                                     |
| > in 2 mM EDTA) using either a 50 ml or 15 ml conical tube (depending |
| > on the volume of cell suspension);                                  |
| >                                                                     |
| > \- centrifuge cells at 1200 rpm for 5 min at 5°C;                   |
| >                                                                     |
| > \- discard supernatant and resuspend cell pellet by finger tapping; |
| >                                                                     |
| > ***[c/p sample preparation:]{.underline}***                         |
| >                                                                     |
| > \- add 5 ml PBS to resuspended cells;                               |
| >                                                                     |
| > \- centrifuge as above;                                             |
| >                                                                     |
| > \- discard supernatant and resuspend cell pellet by finger tapping; |
| >                                                                     |
| > \- add 5 ml 0.075 M KCl and incubate for 8 min at r. t.             |
| >                                                                     |
| > \- centrifuge as above;                                             |
| >                                                                     |
| > \- discard supernatant **without re-suspending the pellet**         |
| >                                                                     |
| > *[(this is crucial pay attention!!!)]{.underline}*;                 |
| >                                                                     |
| > \- fix cells in the pellet with about 2 ml of MeOH : GAA 3 : 1 (v : |
| > v) using a Pasteur pipette, fix for 5 min at r. t.;                 |
| >                                                                     |
| > \- add more fixative so to have a clear milky cell suspension;      |
|                                                                       |
| \- prepare slides spreading fixed cells on a previously moisturized   |
| (in fresh cold distilled                                              |
|                                                                       |
| water) microscope slide;                                              |
|                                                                       |
| > \- make 4 slides *per* group;                                       |
| >                                                                     |
| > \- store slides in microscope slide boxes                           |
| >                                                                     |
| > \- store box at r. t.                                               |
|                                                                       |
| **[RT-PCR SAMPLE PREPARATION]{.underline}**                           |
|                                                                       |
| > ***[RNA Isolation:]{.underline}***                                  |
| >                                                                     |
| > Homogenization with Qiashredder                                     |
|                                                                       |
| -   Collect appropriate number of cells by centrifugation at 1200     |
|     rpm, 4ºC, 5 min. , (one T25 should yield around 50 µg RNA)        |
|                                                                       |
| > \- Decant supernatant and blot edge of tube on paper towel to       |
| > remove residual                                                     |
| >                                                                     |
| > medium                                                              |
| >                                                                     |
| > \- Lyse cells in 350 -- 600 µl Buffer RLT + β-mercaptoethanol by    |
| > pipetting                                                           |
| >                                                                     |
| > (add 10 µl ß-mercaptoethanol per 1 ml Buffer RLT, stable for 1      |
| > month at room                                                       |
| >                                                                     |
| > temp.)                                                              |
| >                                                                     |
| > \- Transfer lysate to a Qiashredder column and centrifuge at top    |
| > speed for 2 min.                                                    |
| >                                                                     |
| > \- Discard column and put a cap on tube                             |
| >                                                                     |
| > \- Store at -80ºC                                                   |
| >                                                                     |
| > (lysates are stable for several months, and can be stored left 20ºC |
| > for shorter                                                         |
|                                                                       |
| term storage \~1 week)                                                |
|                                                                       |
| **[SDS-PAGE ELECTROPHORESIS/WESTERN BLOTTING SAMPLE                   |
| PREPARATION]{.underline}**                                            |
|                                                                       |
| > ***[Protein Isolation:]{.underline}***                              |
| >                                                                     |
| > \- Thaw protease inhibitors and DTT on ice and make fresh lysis     |
| > buffer                                                              |
|                                                                       |
| 1X Lysis Buffer Recipe -- make fresh                                  |
|                                                                       |
|                                                                       |
|  ---------------------- ---------------------- ---------------------- |
|   Component              Storage                Volume (µl) per 1 ml  |
|                                                 buffer                |
|                                                                       |
|   2X WE16 Buffer         4º                     500                   |
|                                                                       |
|   0.5 M DTT              -20º                   2                     |
|                                                                       |
|   20 mg/ml Pefabloc      -20º                   25                    |
|                                                                       |
|   1 mg/ml Leupeptin      -20º                   25                    |
|                                                                       |
|   1 mg/ml Pepstatin      -20º                   10                    |
|                                                                       |
|   1 mg/ml Aprotinin      -20º                   10                    |
|                                                                       |
|   1M ß- glycerophosphate 4º                     80                    |
|                                                                       |
|   0.1 M Na~3~VO~4~       4º                     5                     |
|                                                                       |
|   1M NaF                 4º                     50                    |
|                                                                       |
|   H~2~O                  n/a                    293                   |
|                                                                       |
|  ---------------------- ---------------------- ---------------------- |
|                                                                       |
| **[2X WE16 Buffer (4ºC)]{.underline}**                                |
|                                                                       |
| 50 mM Tris-HCl pH 7.5                                                 |
|                                                                       |
| 250 mM NaCl                                                           |
|                                                                       |
| 5 mM EDTA                                                             |
|                                                                       |
| 0.1% SDS                                                              |
|                                                                       |
| 1% NP40                                                               |
|                                                                       |
| 1% deoxycholic acid                                                   |
|                                                                       |
| 20% glycerol                                                          |
|                                                                       |
| > \- Detach cells collecting media and PBS,                           |
| >                                                                     |
| > \- Centrifuge at 1200Xrpm, 5º, 5 min.                               |
| >                                                                     |
| > \- Resuspend pellet in 5 ml COLD 1X PBS by pipetting up and down 3  |
| > times                                                               |
| >                                                                     |
| > \- Centrifuge again as above                                        |
| >                                                                     |
| > \- Pour off PBS and invert tube on paper towel to remove as much    |
| > residual PBS as                                                     |
|                                                                       |
| possible                                                              |
|                                                                       |
| > \- Resuspend pellet in 200 µl freshly made lysis buffer per 2-3 x   |
| > 10^6^ cells, scale                                                  |
|                                                                       |
| accordingly (save leftover lysis buffer for Bradford assay, store the |
| leftover at                                                           |
|                                                                       |
| > -20°C, when ready to process thaw on ice before centrifugation)     |
| >                                                                     |
| > \- Transfer lysate to 1.5 ml Eppendorf tube                         |
| >                                                                     |
| > \- Centrifuge in Eppendorf centrifuge at 14K rpm, 15'               |
| >                                                                     |
| > \- Remove supernatant to a clean 1.5 ml Eppendorf tube              |
| >                                                                     |
| > \- Store at -80ºC                                                   |
|                                                                       |
| ![](media/image1.png)                                                 |
+-----------------------------------------------------------------------+
| **We will bring compounds already dissolved in the appropriate        |
| buffers.**                                                            |
|                                                                       |
| **The majority of the chemicals used in the protocols reported above  |
| are not toxic. The toxic chemicals we will use/bring are the          |
| following: MeOH (methanol), GAA (Glacial acetic acid),                |
| β-mercaptoethanol, DTT (dithiotheitol), Na~3~VO~4~ (sodium            |
| orthovanadate), NaF (sodium floride),**                               |
|                                                                       |
| **DMSO  |
| (dimethyl sulfoxide), BrdU (Bromodeoxyuridine), HCl (hydrochloric     |
| acid).**                                                              |
|                                                                       |
| **We are not using radioactive materials.**                           |
|                                                                       |
| **We will not use hazardous procedure.**                              |
|                                                                       |
| **The bio-hazardous materials we will bring are the cell lines to be  |
| used in the irradiation experiments. The cell lines we will bring are |
| as follows: mouse embryonic fibroblasts (MEF12(1)), mouse embryonic   |
| fibroblasts (MEF10(1)), human normal lung fibroblasts (MRC5), human   |
| normal skin fibroblasts (CCD32Sk), human bronchial/tracheal           |
| epithelium (NHBE).**                                                  |
|                                                                       |
| **Waste: we will generate only plastic-ware and residual culture      |
| medium. Culture flasks, centrifuge tubes and pipettes will be treated |
| with 10% sodium hypochlorite (bleach) before disposal according to    |
| BNL procedures. Culture Media will be neutralized with 10% sodium     |
| hypochlorite before disposal according to BNL procedures. All the     |
| other solutions will be prepared at appropriate volumes for sample    |
| preparations so that there will not be residuals (see procedures for  |
| sample preparations above).**                                         |
|                                                                       |
| **Shipping the samples back to Iowa City:**                           |
|                                                                       |
| **Samples preparations of microscope slides will be shipped at room   |
| temperature,**                                                        |
|                                                                       |
| **FCM samples will be shipped at 4°C as cell pellet fixed in 95%      |
| ethanol,**                                                            |
|                                                                       |
| **Lysate from cell samples for SDS-PAGE/Western Blotting will be      |
| shipped in dry ice,**                                                 |
|                                                                       |
| **Lysate from cell samples for RT-PCR will be shipped in dry ice,**   |
|                                                                       |
| **Pelleted frozen cells for mutation experiments and live cell        |
| imaging will be shipped in dry ice**                                  |
+-----------------------------------------------------------------------+

6.  ***MATERIALS TO BE USED Please use only the minimum quantity and the
    least hazardous chemicals available to do your experiment
    successfully:***

**Please, refer to point 5 above.**

***6a) Chemicals: List all chemicals including toxic and experimental
substances, controlled substances, compressed gases, and cryogens:***

+----------+---------+----------+-----------+--------+--------------+
| **Name   | **Qua   | *        | *         | **     | **Special    |
| of       | ntity** | *Storage | *Location | Return | Handling**   |
| Ch       |         | Location | of use    | to     |              |
| emical** |         | (        | (Medical, | Home   | **Re         |
|          |         | Medical, | NSRL      | In     | quirements** |
|          |         | NSRL,    | B         | stitut |              |
|          |         | Bi       | iology)** | ion?** |              |
|          |         | ology)** |           |        |              |
+----------+---------+----------+-----------+--------+--------------+
| Eagle's  | 40      | Medical  | Medical   | will   | none         |
| Minimal  | liters  |          | and NSRL  | all be |              |
| E        |         |          |           | used,  |              |
| ssential |         |          |           | if     |              |
| Medium   |         |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| B        | 40      |          | Medical   | will   | none         |
| ronchial | liters  |          | and NSRL  | all be |              |
| Ep       |         |          |           | used,  |              |
| ithelial |         |          |           | if     |              |
| Growth   |         |          |           | left   |              |
| Medium   |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| Du       | 40      | Medical  | Medical   | will   | none         |
| lbecco's | liters  |          | and NSRL  | all be |              |
| Modified |         |          |           | used,  |              |
| Eagle's  |         |          |           | if     |              |
| Medium   |         |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| Pen      | 200 ml  | Medical  | Medical   | will   | none         |
| icillin/ | for 40  |          | and NSRL  | all be |              |
| stre     | liters  |          |           | used,  |              |
| ptomycin | of      |          |           | if     |              |
|          | media   |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| NEAA     | 400 ml  | Medical  | Medical   | will   | none         |
|          | for 40  |          | and NSRL  | all be |              |
|          | liters  |          |           | used,  |              |
|          | of      |          |           | if     |              |
|          | media   |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| fetal    | 4       | Medical  | Medical   | will   | none         |
| bovine   | liters  |          | and NSRL  | all be |              |
| serum    | for 40  |          |           | used,  |              |
|          | liters  |          |           | if     |              |
|          | of      |          |           | left   |              |
|          | media   |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| 1x PBS   | 40      | Medical  | Medical   | will   | none         |
|          | liters  |          | and NSRL  | all be |              |
|          |         |          |           | used,  |              |
|          |         |          |           | if     |              |
|          |         |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| 2 mM     | 4       | Medical  | Medical   | will   | none         |
| EDTA     | liters  |          | and NSRL  | all be |              |
|          |         |          |           | used,  |              |
|          |         |          |           | if     |              |
|          |         |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| 0.075 M  | 2.4     | Medical  | Medical   | will   | none         |
| KCl      | liters  |          | and NSRL  | all be |              |
|          |         |          |           | used,  |              |
|          |         |          |           | if     |              |
|          |         |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| MeOH     | 2       | Medical  | Medical   | will   | none         |
|          | liters  |          | and NSRL  | all be |              |
|          |         |          |           | used,  |              |
|          |         |          |           | if     |              |
|          |         |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| GAA      | 2       | Medical  | Medical   | will   | none         |
|          | liters  |          | and NSRL  | all be |              |
|          |         |          |           | used,  |              |
|          |         |          |           | if     |              |
|          |         |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| RLT      | 240 ml  | Medical  | Medical   | will   | none         |
| buffer   |         |          | and NSRL  | all be |              |
|          |         |          |           | used,  |              |
|          |         |          |           | if     |              |
|          |         |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| β-       | 2.4 ml  | Medical  | Medical   | will   | none         |
| mercapt  |         |          | and NSRL  | all be |              |
| oethanol |         |          |           | used,  |              |
|          |         |          |           | if     |              |
|          |         |          |           | left   |              |
|          |         |          |           | over   |              |
|          |         |          |           | then   |              |
|          |         |          |           | YES    |              |
+----------+---------+----------+-----------+--------+--------------+
| Lysis    | 100 ml  | Medical  | Medical   | will   | none         |
| Buffer   |         |          | and NSRL  | all be |              |
| for      | this    |          |           | used,  |              |
| Western  | buffer  |          |           | if     |              |
| Blotting | co      |          |           | left   |              |
| samples  | ntains: |          |           | over   |              |
|          |         |          |           | then   |              |
|          | WE16    |          |           | YES    |              |
|          | B       |          |           |        |              |
|          | uffer\* |          |           |        |              |
|          | (50 ml) |          |           |        |              |
|          |         |          |           |        |              |
|          | DTT     |          |           |        |              |
|          | (0.2    |          |           |        |              |
|          | ml)     |          |           |        |              |
|          |         |          |           |        |              |
|          | P       |          |           |        |              |
|          | efabloc |          |           |        |              |
|          | (2.5    |          |           |        |              |
|          | ml)     |          |           |        |              |
|          |         |          |           |        |              |
|          | Le      |          |           |        |              |
|          | upeptin |          |           |        |              |
|          | (2.5    |          |           |        |              |
|          | ml)     |          |           |        |              |
|          |         |          |           |        |              |
|          | Pe      |          |           |        |              |
|          | pstatin |          |           |        |              |
|          | (1 ml)  |          |           |        |              |
|          |         |          |           |        |              |
|          | Ap      |          |           |        |              |
|          | rotinin |          |           |        |              |
|          | (1 ml)  |          |           |        |              |
|          |         |          |           |        |              |
|          | Gl      |          |           |        |              |
|          | yceroph |          |           |        |              |
|          | osphate |          |           |        |              |
|          |         |          |           |        |              |
|          | (8 ml)  |          |           |        |              |
|          |         |          |           |        |              |
|          | Na~     |          |           |        |              |
|          | 3~VO~4~ |          |           |        |              |
|          | (0.5    |          |           |        |              |
|          | ml)     |          |           |        |              |
|          |         |          |           |        |              |
|          | NaF (5  |          |           |        |              |
|          | ml)     |          |           |        |              |
|          |         |          |           |        |              |
|          | H~2~0   |          |           |        |              |
|          |         |          |           |        |              |
|          | \*WE16  |          |           |        |              |
|          | Buffer  |          |           |        |              |
|          | co      |          |           |        |              |
|          | ntains: |          |           |        |              |
|          |         |          |           |        |              |
|          | 50 mM   |          |           |        |              |
|          | T       |          |           |        |              |
|          | ris-HCl |          |           |        |              |
|          |         |          |           |        |              |
|          | 250 mM  |          |           |        |              |
|          | NaCl    |          |           |        |              |
|          |         |          |           |        |              |
|          | 5 mM    |          |           |        |              |
|          | EDTA    |          |           |        |              |
|          |         |          |           |        |              |
|          | 0.1%    |          |           |        |              |
|          | SDS     |          |           |        |              |
|          |         |          |           |        |              |
|          | 1% NP40 |          |           |        |              |
|          |         |          |           |        |              |
|          | 1%      |          |           |        |              |
|          | deox    |          |           |        |              |
|          | ycholic |          |           |        |              |
|          | acid    |          |           |        |              |
|          |         |          |           |        |              |
|          | 20%     |          |           |        |              |
|          | g       |          |           |        |              |
|          | lycerol |          |           |        |              |
+----------+---------+----------+-----------+--------+--------------+
| Freezing | 800 ml  | Medical  | Medical   | will   | none         |
| solution |         |          | and NSRL  | all be |              |
|          | s       |          |           | used,  |              |
|          | olution |          |           | if     |              |
|          | co      |          |           | left   |              |
|          | ntains: |          |           | over   |              |
|          |         |          |           | then   |              |
|          | 5 mM    |          |           | YES    |              |
|          | K~      |          |           |        |              |
|          | 2~PO~4~ |          |           |        |              |
|          |         |          |           |        |              |
|          | 25 mM   |          |           |        |              |
|          | KOH     |          |           |        |              |
|          |         |          |           |        |              |
|          | 30 mM   |          |           |        |              |
|          | NaCl    |          |           |        |              |
|          |         |          |           |        |              |
|          | 20 mM   |          |           |        |              |
|          | L(+)    |          |           |        |              |
|          | lactic  |          |           |        |              |
|          | acid    |          |           |        |              |
|          |         |          |           |        |              |
|          | 5 mM    |          |           |        |              |
|          | d       |          |           |        |              |
|          | extrose |          |           |        |              |
|          |         |          |           |        |              |
|          | 0.5 mM  |          |           |        |              |
|          | MgCl~2~ |          |           |        |              |
|          |         |          |           |        |              |
|          | 0.2 M   |          |           |        |              |
|          | s       |          |           |        |              |
|          | orbitol |          |           |        |              |
|          |         |          |           |        |              |
|          | 20%     |          |           |        |              |
|          | DMSO    |          |           |        |              |
+----------+---------+----------+-----------+--------+--------------+

*6b) BIOLOGICALS: List all animals, cell lines (and note if they are
primary or not), blood or body fluids, viruses, viable bacteria, or
toxins of biological origin. Please list detailed description of
organisms below. You may need Institutional Animal Care and Use
Committee (IACUC), Institutional Biosafety Committee (IBC) and/or
Institutional Review Board (IRB) Approval:*

  -------------------- --------------------------- --------------- -------------------
  **Name of            **Description**             **Disposal      **Special
  Biological**                                     method at BNL   Handling/Approval
                                                   or note return  (IRB, IBC)**
                                                   to Home         
                                                   Institution**   

  mouse embryonic      Cell line                   Residual cells  N/A
  fibroblasts                                      will be         
  (MEF12(1)                                        neutralized     
                                                   with 10% sodium 
                                                   hypochlorite    
                                                   before          
                                                   disposal.       
                                                   Residual        
                                                   culture media   
                                                   will be         
                                                   neutralized     
                                                   with 10% sodium 
                                                   hypochlorite    
                                                   before          
                                                   disposal.       

  mouse embryonic      Cell line                   As above        N/A
  fibroblasts                                                      
  (MEF10(1)                                                        

  human normal lung    Cell line                   As above        N/A
  fibroblasts (MRC5)                                               

  human normal skin    Cell line                   As above        N/A
  fibroblasts                                                      
  (CCD32Sk)                                                        

  human                Cell line                   As above        N/A
  bronchial/tracheal                                               
  epithelium (NHBE)                                                
  -------------------- --------------------------- --------------- -------------------

*6c) Radioactive Materials: List any radioactive materials, include any
handling of activated materials:*

+-------+------+------------+---------+-------------+----------------+
| **N   | **Qu | **Storage  | **L     | **Disposal  | **Special      |
| ame** | anti | Location   | ocation | method at   | Handling**     |
|       | ty** | (Medical,  | of use  | BNL or note |                |
|       |      | NSRL,      | (M      | return to   | **             |
|       |      | Biology)** | edical, | Home        | Requirements** |
|       |      |            | NSRL    | In          |                |
|       |      |            | Bio     | stitution** |                |
|       |      |            | logy)** |             |                |
+-------+------+------------+---------+-------------+----------------+
|       |      |            |         |             |                |
+-------+------+------------+---------+-------------+----------------+
|       |      |            |         |             |                |
+-------+------+------------+---------+-------------+----------------+

> ***If you are required to handle materials shortly after beam exposure
> (activated materials) indicate time frame required:***

  --------------- -------------------------------------------------------
  **Time          **Description**
  Required**      

  **Soon as the   **We will need to collect zero time points immediately
  beam is off --  after irradiation. Next time points will be at 8 h
  would need 30   post-irradiation.**
  minutes per     
  beam**          
  --------------- -------------------------------------------------------

**7. *EQUIPMENT: List any equipment you will bring to BNL. Please list
where you will use it (NSRL, Medical, Biology). Listing of Potentially
Hazardous Equipment Must Include: electric equipment not UL approved or
certified to meet National Electrical Code, electronic equipment,
detectors with flammable gases and flammable gas targets,
flammable-combustible (e.g. plastic detector materials), samples,
reactive metals etc, lasers, ovens, pumps, cryostats, pressure devices
or pressure vessels, vacuum windows or vacuum vessels, liquid or gas
mixing or containment systems, UV lamps, high-temperature devices,
material handling devices, solenoids, spectrometer magnets, structures
supporting heavy loads, compressed air or gas systems, RF or microwave
devices, sound systems or noise greater than 85 dBA, items that emit
liquids, gases, or vapors from the experiment, welding or burning tools,
or any equipment or activities that require special written procedures
by the User.***

+-----------------------------------------------------------------------+
| **Description**:                                                      |
|                                                                       |
| **No equipment will be brought to BNL**                               |
+-----------------------------------------------------------------------+

**8. WASTES: (including clean waste, hazardous waste, radioactive waste,
medical/biohazard waste.)**

+----------+----------+------------------+----------------------------+
| **Name   | **Descr  | **Anticipated    | **Disposal Method**        |
| of       | iption** | quantity**       |                            |
| Waste**  |          |                  |                            |
+----------+----------+------------------+----------------------------+
| Plas     | Culture  | 1200 flasks      | Residual media in flasks   |
| tic-ware | flasks   |                  | will be neutralized with   |
|          |          | 2000 pipettes    | 10% sodium hypochlorite    |
|          | Pipettes |                  | and disposed of according  |
|          |          |                  | to BNL procedures          |
+----------+----------+------------------+----------------------------+
| Mouse    | Residual | Few hundred of   | Residual cells will be     |
| and      | from     | ml (cells will   | neutralized with 10%       |
| human    | cell     | mostly all be    | sodium hypochlorite and    |
| (not     | passages | used, not        | disposed of according to   |
| primary) |          | disposed of)     | BNL procedures             |
| cells    |          |                  |                            |
+----------+----------+------------------+----------------------------+

**9. USER COMMENTS:**

+-----------------------------------------------------------------------+
| **I have already completed all the required web-training**            |
|                                                                       |
| **I was at BNL for on-site training November 8^th^, 2006**            |
+-----------------------------------------------------------------------+

**10. *TRAINING: Training requirements for each experiment will be
posted on the NASA website. Listed below are the specific training
requirements based on the work to be performed.***

**For return users, you may check your training status on-line at**
[http://training.bnl.gov](http://training.bnl.gov/) **to see which
courses you need to complete again.**

**All NSRL Users must complete the following 4 courses:**

1.  **C-A Radiobiology Users Training**

-   Initial training - Classroom training necessary - contact
    userscenter@bnl.gov for schedule.

-   Renewals- every 24 months, complete Challenge exam at
    [http://training.bnl.gov](http://training.bnl.gov/) (study guide is
    available), or repeat classroom training.

1.  **Radiological Worker 1**

-   Part 1 can be done on-line at http://training.bnl.gov

-   Part 2 consists of a challenge exam to be taken at BNL. Study guide
    is at http://training.bnl.gov

> (Note: If you are from another DOE facility you can apply for DOE
> Radiation Worker Reciprocity by completing [exemption
> form](http://training.bnl.gov/)**).**

2.  **Cyber Security online at**
    [http://training.bnl.gov](http://training.bnl.gov/)**.** Initial
    training only (no requalification required)

3.  **Guest Site Orientation online at**
    [http://training.bnl.gov](http://training.bnl.gov/)**.** Initial
    training only (no requalification required)

**In addition, ALL participants using laboratory facilities in the
Medical or Biology Departments must complete the following.**

**If you are using chemicals, as noted in this experimental safety
review, you must complete the following on-line courses**

-   Laboratory Standard (required every two years)

```{=html}
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```
-   Hazardous Waste Generator (required annually)

-   Regulated Medical Waste Generator Training (initial training only)

**If you are using Human blood, tissues, or primary human cells:**

-   Bloodborne Pathogens Training (required annually)

**If you are using cryogens (liquid nitrogen or helium), you must
complete the following on-line course:**

-   Cryogen Safety Awareness (initial training only)

**If you are using compressed gases, you must complete the following
on-line course:**

-   Compressed Gas Safety (initial training only)

**If you are using dispersible Radioactive Materials such as tagged
cells, or will handle your samples while activated from beam exposure,
you must complete the following:**

-   Radioactive Waste Generator (HP-RADIGEN) available on-line

-   Benchtop Dispersibles Training (required every two years) - contact
    Ann Emrick, <emrick@bnl.gov>, to arrange for course)

**If you are using Animals:**

-   Laboratory Animal Training (LAT I): will be administered upon
    arrival. (Initial training only, no requalification)

**If you are using Controlled Substances:**

-   Controlled Substance Awareness

-   DEA Background Check: Contact Medical Chairman's Office at
    (631)344-3715

> **ALL USERS MUST READ AND SIGN THE [LOW HAZARD-SKILL OF THE CRAFT WORK
> PLAN]{.underline} SPECIFIC FOR THE EXPERIMENTAL RUN THEY ARE
> ATTENDING**
>
> **EXPERIMENTAL SPOKESPERSON -- PRINCIPAL INVESTIGATOR, PLEASE SIGN AND
> DATE BELOW:**
>
> SIGNATURE:\_\_\_Fiorenza Ianzini Nivember 14,
> 2006\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

  ---------------------------------- --------------------------------------------------
  ***Life Sciences Experimental        
  Review Committee Designee                                                          
  Approval:***                       

  ***C-A Experimental Safety Review  
  Committee Designee Approval:***    

  ***Environmental Safety &Health    
  Staff Comments and                 
  Requirements:***                   

  ***Approval Date***                
  ---------------------------------- --------------------------------------------------