eamag/protocols-with-steps huggingface dataset:
TITLE: Immunocytochemistry Staining for Methanol Fixed Cells
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Sterilzation:]
Transfer a single cover slip into a 12-well plate, then add 1 mL of 70% Ethanol into a well for 20 minutes at room temperature.
?. [Sterilzation:]
Wash quickly three times with PBS.
?. [Poly-Lysine Coating for 12-Well Plates (optional; for loosely attached cells):]
Add 1 mL of 0.1 mg/mL Poly-D-lysine solution into a well for 15 minutes at room temperature.
?. [Poly-Lysine Coating for 12-Well Plates (optional; for loosely attached cells):]
Wash quickly three times with PBS and let dry before plating cells.
?. [Sample Preparation:]
Grow cultured cells on cover slips or in wells overnight at 37°C. At the time of fixation, cells should be ~70-80% confluent in single layer.
?. [Sample Preparation:]
Rinse cells briefly in PBS.
?. [Sample Preparation:]
Fix and permeabilize cells by incubation with cold 100% methanol for 5-15 minutes at -20°C.
?. [Sample Preparation:]
Rinse three times quickly in PBS.
?. [Sample Blocking:]
Block samples in 1 mL of blocking buffer at room temperature for 30 minutes.
?. [Sample Staining:]
Dilute the primary antibody to the recommended concentration/dilution in blocking buffer.
?. [Sample Staining:]
For 8-well chamber slides, add 200 µL per well. For 12-well plates, add 500 µL per well. Incubate two to three hours at room temperature or overnight at 4°C. If using conjugated antibodies, perform this step in the dark.
?. [Sample Staining:]
For surface staining, rinse 3 times quickly in PBS. For intracellular staining, quickly wash once followed by incubation with wash buffer for 5-10 minutes. Then quickly wash additional two times.Note: If using primary antibodies directly conjugated to fluorochromes, then skip to step 12.
?. [Sample Staining:]
Prepare fluorochrome-conjugated secondary antibody in blocking buffer according to the manufacturer’s specification data sheet, and add 200 µl per well to the 8-well chamber slides. For 12-well plates, add 500 µL per well.
?. [Sample Staining:]
Incubate the samples for one hour, at room temperature, in the dark.
?. [Sample Staining:]
For surface staining, rinse three times quickly in PBS. For intracellular staining, quickly wash once followed by incubation with wash buffer for 5-10 minutes, then quickly wash additional two times.
?. [Sample Staining:]
Optional: To stain F-actin, prepare a working solution of Flash Phalloidin™ by diluting it 1:20-1:100 in PBS. Add 200 µL per well for an 8-well plate or 500 µL per well for a 12-well plate. Stain for 20 minutes at room temperature in the dark.
?. [Sample Staining:]
Apply anti-fade mounting medium to the cover slip.
?. [Sample Staining:]
Seal slides with nail polish.
TITLE: Inclusion and Exclusion criteria 
AUTHORS: pooja.devrukhkar, hannah.anvari, asma.giornazi Asma Giornazi, shriya.shah, maryellen.pavone, francesca.e.duncan francesca.duncan

[DESCRIPTION]
The purpose of this protocol is to describe the inclusion/exclusion criteria for collection of human follicular fluid, ovarian tissue, and matched biofluids (plasma and urine) for research. 

[GUIDELINES]

Researchers will adhere to all safety and training protocols required by the institution including but not limited to: 

1. Biosafety Certification 

2. Bloodborne Pathogens Certification 

3. Collaborative Institutional Training Initiative (CITI program) certification 




[STEPS]
SECTION: Collection of Research Specimens for U54/UG3 – Done by Pathology
1. Human ovaries and follicular fluid are obtained following informed consent from the Northwestern University Reproductive Tissue Library (NU-RTL).



SECTION: Collection of Research Specimens for U54/UG3 – Done by Pathology
2. NU-RTL uses an established IRB-approved protocol that facilitates the acquisition of de-identified
human reproductive specimens including but not limited to gynecologic tissue (ovaries) and follicular fluid for research purposes.  



SECTION: Collection of Research Specimens for U54/UG3 – Done by Pathology
3. Blood and urine can also be collected at the time of consent or procedure, and basic de-identified demographic and medical health information is also maintained.  



SECTION: Study-Specific Inclusion/Exclusion Criteria:
4. Inclusion Criteria
SECTION: Study-Specific Inclusion/Exclusion Criteria:
4.1. 

a.     aged 18 and older (no upper age limit)


b.    for ovarian tissue, women undergoing bilateral salpingo-oophorectomies (BSOs), with the absence of any ovarian neoplasia, as well as hysterectomies (along with Bilateral Salpingo- Oophorectomies) for conditions including but not limited to endometrial hyperplasia and prolapse where no ovarian neoplasia is identified.


c.     for follicular fluid, women undergoing ART procedures for egg donation, male factor infertility, elective oocyte vitrification, and preimplantation genetic diagnosis for single gene disorders.



SECTION: Study-Specific Inclusion/Exclusion Criteria:
5. Exclusion Criteria
SECTION: Study-Specific Inclusion/Exclusion Criteria:
5.1. Women with endometriosis diagnosis
TITLE: Mouse perfusions and brain tissue processing
AUTHORS: Ayse Ulusoy, Shirley Lee, Angela Rollar, Michael Helwig, Michael Klinkenberg, Sinead O'Sullivan, Rita Pinto-Costa, Donato Di Monte

[DESCRIPTION]
The protocol describes tissue processing for histological analyses and includes perfusions, brain fixation, and tissue harvesting from perfused rodents. 

[STEPS]
1. Inject sodium pentobarbital (600 mg/kg, i.p.) to sacrifice the mouse
2. Once the respiration is stopped, open the chest and place the cannula connected to a peristaltic pump into the heart. Make sure that the cannula has access to the ascending aorta. 

3. Perfuse, first with 20 ml saline solution, kept at room temperature, and then with ice-cold 60 ml 4% (w/v) paraformaldehyde.

4. Quickly remove the brain and immerse in 4% paraformaldehyde for 24 hours. 
5. For cryopreservation, place the brain in a 30% (w/v) sucrose solution. 
6. Once sunk, section the brain on the coronal plane (35 µm) using a freezing microtome.
TITLE: Structural prediction of VPS13C with AlphaFold2
AUTHORS: Shujun  Cai, Pietro  De Camilli

[DESCRIPTION]
This protocol describes the procedure of structural prediction of full-length human VPS13C and its truncation mutant with AlphaFold2 and the procedure to combine each segments into one structure. 

[STEPS]
SECTION: Structure prediction
1. 
 Separate Full-length VPS13C and truncation mutant into three pieces and two pieces, respectively, i.e., a.a. 1-1860, 1201-2340, 1801-3753 for full-length VPS13C and a.a. 1-1762, 1277-3240 for VPS13CΔ1235-1748.
 

SECTION: Structure prediction
2. Install AlphaFold 2.0 on the Yale Farnam high performance computer cluster, used for structural prediction of each segment. 
 

SECTION: Combination of segments
3. Open UCSF ChimeraX software and import pdb files of segments.
SECTION: Combination of segments
4. Align the fragments with overlapping regions with “mmaker” command. 
SECTION: Combination of segments
5. Delete the overlapping regions from adjacent fragments with “delete” command. 
SECTION: Combination of segments
6. At the connection point, form a carbon–nitrogen bond  with “build join peptide” command to connect two amino acid residues from two fragments.
TITLE: Protocol for Mouse IP Anesthesia (Ketamine-Xylazine)
AUTHORS: Haowei Li, Markus Holzl, Mohsen Khosravi-Maharlooei, Nichole Danzl, Austin Chen, Megan Sykes

[DESCRIPTION]
This protocol details the constitution of our anesthesia cocktail that we use for sedating our mice when performing surgeries. The surgeries we typically perform are thymectomies and implantation of material under the kidney capsule (through open abdomen or minimally invasive approaches).

 


 


[STEPS]
SECTION: For Intraperitoneal (IP) Approach:
1. Use 10 uL/gram of anesthesia

Comments:
1. It takes time until the mice fall asleep 

2. Very often, mice do not fall asleep but move around like zombies. This requires another small dose of IP ketamine-xylazine injection or a small dose of IV injection

a. When doing IP injection again, we have injected IP with a volume of about 1/3 of original volume

b.  For IV approach after IP, it takes very little anesthesia for them to fall asleep. We usually use around 10-20 uL. Inject this slowly into the mouse. The mouse should fall asleep rapidly. It is very easy to overdose with IV injection so be careful.
TITLE: Infection of Biomphalaria glabrata snails with Schistosoma mansoni miracidia
AUTHORS: Sarah K Buddenborg

[DESCRIPTION]
To infect Biomphalaria glabrata snails with miracidia hatched from Schistosoma mansoni eggs

[STEPS]
SECTION: Liver preparation
1. Collect livers from patent mice into 50ml Falcon tubes containing pre-warmed 37°C 1x DPBS
SECTION: Liver preparation
2. Remove livers from PBS and place in large mortar or laboratory blender
SECTION: Liver preparation
3. Gently homogenise liver tissue
SECTION: Liver preparation
4. Place homogenised liver slurry into a flask (size of flask depends on how many livers you have) and fill with diH2O or aquarium water
SECTION: Hatching miracidia
5. Cover flask in aluminum foil and shine light horizontally across the opening of the flask for 1-2 hrs
SECTION: Hatching miracidia
6. Take a small aliquot of water from the top of the flask using a glass Pasteur pipette and place into a petri dish. Using a stereomicroscope, check for swimming miracidia
SECTION: Exposing snails to miracidia for regular life cycle maintenance
7. Under a microscope, collect 15 miracidia with a glass Pasteur pipette per well

Alternatively (but less preferred) estimate the number of miracidia by counting 12-5µl aliquots in a petri dish. After collecting (shaking the tube in between) and placing the aliquots in the petri dish add 5µl of Lugol to each drop (this kills and stains the miracidia) 
SECTION: Exposing snails to miracidia for regular life cycle maintenance
8. Place individual 5-8mm snails into 24-well culture plates and cover snails completely with 1x aquarium water
SECTION: Exposing snails to miracidia for regular life cycle maintenance
9. Expose snails to miracidia for at least 3 hours (up to overnight)
SECTION: Exposing snails to miracidia for regular life cycle maintenance
10. After exposure, remove the snails carefully using featherweight forceps and put them in a new tank with food
SECTION: Exposing snails to miracidia for monomiracidium infections
11. Dilute miracidia so that one miracidium can easily be collected in ~3-5µl of water
SECTION: Exposing snails to miracidia for monomiracidium infections
12. Collected single miracidium using fresh 10µl pipette each collection and place in 24-well plate
SECTION: Exposing snails to miracidia for monomiracidium infections
13. After a plate is filled, check each well under a microscope to verify there is a single miracidium in each well
SECTION: Exposing snails to miracidia for monomiracidium infections
14. Place individual 5-8mm snails in the wells containing confirmed single miracidium and cover snails completely with 1x aquarium water
SECTION: Exposing snails to miracidia for monomiracidium infections
15. Leave the plate overnight (inside incubator or room at 28°C)
SECTION: Exposing snails to miracidia for monomiracidium infections
16. The following day, transfer the snails to a new tank with food
TITLE: Native-PAGE analysis of VCP hexamer
AUTHORS: Itika Saha, F. Ulrich Hartl, Mark S. Hipp

[DESCRIPTION]
Valosin-containing protein (VCP) is a homo-hexameric AAA+ ATPase in eukaryotic cells. This protocol describes the analysis of myc-tagged versions of VCP transiently transfected in HEK293 cells (stably expressing and propagating aggregates of Tau repeat domain fused to YFP) for hexamer formation.

[STEPS]
1. Plate 1.5x105 cells in 12-well plate.
2. Next day, transfect with plasmids expressing myc-tagged VCP variants (Saha et al. BioRxiv, 2022) using a standard transfection protocol.
3. Two days later, collect cells and lyse them in 50 µL 0.5% Triton X-100/PBS supplemented with protease inhibitor cocktail (Roche) and DNase for 1 h on ice.
4. Centrifuge lysates at 10,000 x g for 2 min and collect supernatant.
5. Determine protein concentration in the supernatant and normalize across all samples.
6. Add 2x native sample buffer (40 % glycerol, 240 mM Tris pH 6.8, 0.04 % bromophenol blue) to 40 µg lysate.
7. Run samples on a Native PAGE gel (e.g. Novex Value 4 to 12% Tris-glycine gels (Thermo)) in 20 mM Tris 200 mM Glycine buffer at pH 8.4.
8. Transfer proteins to nitrocellulose membrane in standard Tris-glycine buffer, block in 5% low-fat dry milk for 1 h at room temperature (RT). 
 

9. Dilute anti-myc (9E10) and anti-VCP (1:2000, Novus Biologicals) primary antibodies together in blocking solution and incubate membrane overnight.
10. Next day, wash membrane 3 times with TBST and incubate with anti-mouse (LI-COR Biosciences Cat# 926-68070, RRID:AB_10956588; 1:10,000) and anti-rabbit (LI-COR Biosciences Cat# 926-32211, RRID:AB_621843; 1:10,000) fluorescent secondary antibodies for 2 h at RT.
11. Wash membrane 3 times with TBST.
12. Detect fluorescent myc and VCP signal on a fluorescent imager.
TITLE: Select, load, annotate, normalize, and process toxicogenomic raw data from GEO and ArrayExpress
AUTHORS: Andreas Schüttler

[DESCRIPTION]
<p>Gene expression databases like Gene Expression Omnibus or ArrayExpress by now contain a wealth of toxicogenomic datasets. This is a offers great possibilities for advanced data analyses, like meta-analyses, co-expression studies, etc.</p>
<p> </p>
<p>However, automated retrieval of this data is still a challenge.</p>
<p> </p>
<p>With this computational pipeline we retrieve toxicogenomic data of the <em>Danio rerio</em> (zebrafish) embryo from GEO and ArrayExpress.</p>
<p>To make the data as comparable as possible, we download the raw datasets, and re-map the probes or probesets to the most recent version.</p>
<p> </p>
<p>In the end a matrix with logFC in response to different chemical treatments is compiled.</p>

[STEPS]
?. [Select data from Gene expression databases]
1. GEO: The first step is to retrieve metadata from Gene Expression Omnibus. This is achieved with the help of the R-package 'GEOmetadb'.From the metadata and from manually curated information, datasets are selected and list for downloading data are created.
?. 2. ArrayExpress: The same as for GEO is done for ArrayExpress.
?. [Download Array and Platform data]
Next step is to download the array and platform data selected in step 1.We create a "data" directory where all data is downloaded to.
?. [Probe mapping]
Since microarrays are designed for different genome version, it is necessary to re-map the probes to the recent genome version (here dRer11).For probe mapping, as a first step, fasta-files have to be created from the downloaded platform-files from GEO and ArrayExpress.
?. [Probe mapping]
Perform Blat
?. [Probe mapping]
map with gene annotation
?. [Create target file and table of comparisons]
Before loading the data, we compile a targets data frame and a table with all comparisons for logFC calculation
?. [Read raw data]
Based on the R-packages "limma" and "oligo", data is loaded into R.
?. [Create logFC matrix]
Last but not least a logFC matrix is created from all normalized data.
TITLE: IPA Core Training Guide
AUTHORS: Courtney Comrie

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol serves as the master plan for new IPA Core trainees. </div><div class = "text-block">For questions on the different trainings reach out to Courtney Comrie.</div></div>

[STEPS]
?. [Gain Access to Linux Boxes]
Speak to Dr. Hutchinson or one of the graduate students about getting access to the linux box.After being added to the linux box access list, go to https://dx.doi.org/10.17504/protocols.io.62whgfe for directions on how to enter the linux box through X2Go Client.
?. [Linux Training]
The lab utilizes softwares that use Linux to be run and called. In order to run many of the image analyis softwares, you must first learn to Linux. Cornell University offers a free and comprehensive virtual workshop for beginners in Linux. Complete the training in the link before moving on: https://cvw.cac.cornell.edu/Linux/
?. Obtain access to the HPC web interface through lab account, and follow the "HPC Account Set Up and Access" protocol to get familiar with the interface.
?. With practice data follow protcols Building Data Structures, DIFFPREP, DRBUDDI, and DRTAMAS in that order.
?. Note: In these protocols you will get exposure to other software besides TORTOISE.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p><strong>Equipment</strong></p>
<p> </p>
<p><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/usmerye.png" width="327" height="224" data-src="https://s3.amazonaws.com/pr-journal/uskerye.png" data-ofn="Bildschirmfoto 2018-03-15 um 20.32.37.png" /></strong></p>
<p><br />- <a href="https://www.sartorius.com/sartorius/en/EUR/Applications/Laboratory/Sample-Preparation/Ultrafiltration/Vivaflow-200/p/M_Vivaflow_200?childprod=VF20P7" target="_blank" rel="noopener noreferrer">Vivaflow Cartridge 0.2 μm</a> (PES) VF20P7</p>
<p>-<a href="https://www.coleparmer.com/i/masterflex-l-s-precision-pump-system-easy-load-ii-head-6-600-rpm-90-260v/7791620" target="_blank" rel="noopener noreferrer"> Masterflex Pump 6-600 rpm</a> (ref Bioblock F39671)</p>
<p>- Rotor 3 “cylinders” (ref F39110)<br />- 1 high throughput head (ref F40103) (can be replaced with quick load head)<br />- Replace tube provided by a stronger tube with two connectors (see picture)<br />- Bottle 6 L<br />- Bottle 1 L<br />- Conical tube 50 mL<br />- Masterflex Tygon tubing size 16<br />- Plastique pipettes (1 mL)<br />- Clamps with a screw (to control retentate1 speed)<br />- Clamps</p>
<p><br /><strong>Solutions</strong></p>
<p><br />- Rinsing solution for regenerated cellulose membranes: 250 ml of 0.1 M NaOH<br />- Storage solution: Deionised water with 10 % ethanol.<br />- Deionized water<br />- Filtered seawater</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Leica SP8 Confocal Imaging
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the basic setup and scanning using the Leica SP8 confocal microscope of fluorescently-labeled mouse brain tissue sections mounted on positively charged 1” x 3” microscope slides.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Preparing_Duckweed-Bacteria_Cultures
AUTHORS: Kenneth Acosta

[DESCRIPTION]
Protocol to generate duckweed-bacteria cultures to study duckweed-bacteria interactions.

[GUIDELINES]
Some bacteria may grow slowly. In this case, more time is needed for bacterial cultures to grow to sufficient concentrations.

Some bacteria do not pellet easily. Longer centrifugation times and higher centrifugation speeds may be needed for these types of bacteria. 

[STEPS]
SECTION: Day 1
1. Inoculate 5 mL liquid cultures from bacterial glycerol stocks.
SECTION: Day 1
2. Grow 5 mL liquid bacterial cultures overnight at 28C at 250 rpm.
SECTION: Day 2
3. Use 500 uL of liquid bacterial culture from Day 1 to inoculate 50 mL liquid cultures.
SECTION: Day 2
4. Grow 50 mL liquid bacterial cultures overnight at 28C at 250 rpm.
SECTION: Day 3 (t = 0 days, start of experiment)
5. Transfer 25 mL of bacterial cultures to sterile 30 mL centrifuge tubes.
SECTION: Day 3 (t = 0 days, start of experiment)
6. Spin at 8000 rpm for 5 min at 4C.
SECTION: Day 3 (t = 0 days, start of experiment)
7. Decant SN.
SECTION: Day 3 (t = 0 days, start of experiment)
8. Add 25 mL of sterile H2O to pellets. Resuspend pellets.
SECTION: Day 3 (t = 0 days, start of experiment)
9. Spin at 8000 rpm for 5 min at 4C.
SECTION: Day 3 (t = 0 days, start of experiment)
10. Decant SN.
SECTION: Day 3 (t = 0 days, start of experiment)
11. Add 25 mL of sterile H2O to pellets. Resuspend pellets.
SECTION: Day 3 (t = 0 days, start of experiment)
12. Spin at 8000 rpm for 5 min at 4C.
SECTION: Day 3 (t = 0 days, start of experiment)
13. Decant SN.
SECTION: Day 3 (t = 0 days, start of experiment)
14. Suspend in 25 mL of media.
SECTION: Day 3 (t = 0 days, start of experiment)
15. Read OD600. In a volume of 50 mL dilute to OD600 = 0.2.
SECTION: Day 3 (t = 0 days, start of experiment)
16. Transfer 50 mL cultures to baby jars.
SECTION: Day 3 (t = 0 days, start of experiment)
17. Inoculate cultures with duckweed to cover the surface of the culture.
TITLE: Taq PCR (Protocol for ZymoTaq™ PreMix)
AUTHORS: Alba Balletbó

[STEPS]
?. Add all components in a 250 μL tube making up to a 25 or 50 μl reaction. If performing various PCR with different templates, a Master Mix is recommended to be done.   ABC1ReagentVolumeFinal Concentration2ZymoTaq™ PreMix25 µL1X3Forward Primer (10 µM)Variable 0.3 to 1 µM 4Reverse Primer (10 µM) Variable 0.3 to 1 µM 5TemplateVariable6ddH2Oto 50 µL7Total Volume50 µL
ABC1ReagentVolumeFinal Concentration2ZymoTaq™ PreMix25 µL1X3Forward Primer (10 µM)Variable 0.3 to 1 µM 4Reverse Primer (10 µM) Variable 0.3 to 1 µM 5TemplateVariable6ddH2Oto 50 µL7Total Volume50 µL
?. Gently mix the PCR reactions and transfer the tubes to a thermocycler. Thermocycling conditions for a routine PCR:  ABCD1StepTemperatureTimeCycles2Initial Denaturation10 minutes13Denaturation94-96ºC30 sec30-404AnnealingVariable30-40 sec5Extension72ºC30 sec - 1 minute*6Final Extension72ºC7 minutes17Hold4ºC> 4 minutes1
ABCD1StepTemperatureTimeCycles2Initial Denaturation10 minutes13Denaturation94-96ºC30 sec30-404AnnealingVariable30-40 sec5Extension72ºC30 sec - 1 minute*6Final Extension72ºC7 minutes17Hold4ºC> 4 minutes1
TITLE: Lentivirus Production
AUTHORS: Yiqin Shen

[DESCRIPTION]
A protocol to produce lentivirus through cell transfection

[STEPS]
SECTION: Day 1
1. Coat 15ml cell culture plates with D-poly-lysine (50ug/ml) for 30 min to60 min . 
Wash plates with cell culture water for 1 to 3 times. Let the plates to air dry.

SECTION: Day 2
3. Change media about 3 hours prior to transfection.
SECTION: Day 3
6. Change media for all of the plates. Add 20 mL fresh media.

SECTION: Day 4
7. Collect media from the plates (~24 hours after media change). Store at 4 degrees. 
SECTION: Day 4
8. Add 20 mL of fresh media.

SECTION: Day 5 &amp; 6
9. Repeat collection as day 4. 
SECTION: Day 1
2. Plate pre-grown low passage HEK cells at 6e^6 cells per plate.
Add 20ml of media. 
Incubate overnight at 37 °C 5% CO2.

 

SECTION: Day 2
4. Transfect cells with viral components using CaCl2 transfection Kit in the afternoon.
SECTION: Day 2
5. Incubate overnight at 37 °C 5% CO2.

SECTION: Day 2
4.1. Each plate = 20/25ug desired plasmid, 10.2ul PMD26, 15.6ul PsPAX, 93 ul CaCl2; add water to 750 ul; 750 ul HBSS
SECTION: Day 2
4.2. Add HBSS to tube 2; add plasmid, CaCl2, H2O to tube 1, and mix by vortex.
SECTION: Day 2
4.3. Add tube 1 to tube 2 (on medium speed vortex) drop by drop (to form droplets).
SECTION: Day 2
4.4. Wait 30 min; vortex again before use.
Add 1.5 mL to each plate, distribute evenly across the plate. 
Rock the plates gently to allow more distribution.
SECTION: Day 5 &amp; 6
10. Spin down collected media at 500 x g, 10 min, 4 °C .

SECTION: Day 5 &amp; 6
11. Add 36ml of supernatant from step 10 to 12 ml of Lenti-X concentrator. Incubate the mixture at 4 °C  in a rocker, for 240 min to 30 min .

SECTION: Day 5 &amp; 6
12. Centrifuge the mixture from step 11 at 1500 x g, 45 min, 4 °C . After centrifugation, an off-white pellet will be visible.

SECTION: Day 5 &amp; 6
13. Remove supernatant from the tubes. 
Resuspend the pellet in 100ul HBSS per pellet. 
Aliquote 200ul per freezing tube. 
Place the tubes on ice or freezing blocks immediately when you are finished, and transfer to -80 °C  for long term storage.