eamag/protocols-with-steps huggingface dataset:
TITLE: CRISPR/Cas9 genome editing
AUTHORS: wusj, schekman

[DESCRIPTION]
This protocol describes the generation of DNAJC5 KO using CRISPR/Cas9 edition

[STEPS]
SECTION: CRISPR/Cas9 genome editing
1. gRNA targeting exon 4 of DNAJC5 ( CACC GGAG GCCG CAGA AGAC AAAC A) was inserted into a
pX330-based plasmid expressing Venus fluorescent protein
SECTION: CRISPR/Cas9 genome editing
2. HEK293T cells were transfected with pX330-pX330-Venus-DNAJC5-Exon4-gRNA by Lipofectamine 2000 followed the commercial protocol by Thermo Fisher.
SECTION: CRISPR/Cas9 genome editing
3. After 48 hr, we diluted the cells and single colonies were isolated from 96-well plate, expanded, and determined for DNAJC5 KO by immunoblot.
TITLE: Tetraspeck Bead Imaging 
AUTHORS: Joseph S Beckwith

[DESCRIPTION]
Protocol for imaging tetraspeck beads on glass coverslips.

[STEPS]
SECTION: Slide Preparation
1. Glass coverslips (Fisher Scientific, 12373128, #1 thickness 22 mm x 50 mm) were plasma cleaned for 30 min (Ar plasma cleaner, PDC-002, Harrick Plasma).
SECTION: Slide Preparation
2. Stick a frame-seal slide chamber (9 mm x 9 mm, SLF0201, Biorad) on the cover glass. Use some blunt tweezers to press down the sticker on the glass.
SECTION: Slide Preparation
3. Add 50 μl of 0.01 % w/v poly-L-lysine (PLL, P4707, Sigma-Aldrich) to the well and wait for 10-20 min.
SECTION: Slide Preparation
4. Use a pipet to remove excess PLL.
SECTION: Slide Preparation
5. Wash with 50 μl of filtered (0.02 μm syringe filter, Whatman, 6809-1102) PBS. Pipet up and down in the corners of the well to wash. Repeat this step 3 times.
SECTION: Slide Preparation
6. Remove excess PBS and add 50 μl of the diluted TetraSpeck (1:625 dilution, 0.1 μm diameter TetraSpeck Microspheres, Thermo Fisher) beads to the well. Wait 2-3 minutes to let the beads settle and attach to the PLL-coated glass.
SECTION: Slide Preparation
7. Remove excess solution using a pipet.
SECTION: Slide Preparation
8. Wash with 50 μl of filtered PBS. Pipet up and down in the corners of the well to wash. Repeat this step 3 times.
SECTION: Slide Preparation
9. Remove excess PBS and add 50 μl filtered PBS to the well. The sample should not dry out!
SECTION: Imaging
10. Image the slide on a light microscope.
TITLE: Artificial Cerebrospinal Fluid IX (ACSF.IX)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Artificial Cerebrospinal Fluid IX (ACSF.IX) is used for applications including tissue bath solution during electrophysiological recording for probing synaptic connections under physiological conditions.</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: MojoSort™ Whole Blood Human CD8 Nanobeads Whole Blood Column Protocol
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with </span><span style = "font-weight:bold;">pre-diluted </span><span>MojoSort™ reagents and using the columns as indicated by the manufacturer.</span></div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service (tech@biolegend.com) if further assistance is needed.</span></div></div>

[STEPS]
?. Collect whole blood in collection tube that has anticoagulant, preferably EDTA. Note: Keep MojoSort™ Buffer on ice throughout the procedure.
?. Add 1mL of whole blood into a new tube.
?. Resuspend the beads by vortexing, maximum speed, 5 touches. Add 50 µL of pre-diluted conjugated Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating larger starting volume of whole blood. For example, add 100 µL of pre-diluted Nanobeads for separating 2mL of whole blood.
?. Proceed to separation on column as indicated by the manufacturer. Note: There are several types of commercially available columns, depending on your application. Choose the one that fits best your experiment.  ABCDE1Max volume of whole bloodColumn rinse volumeCell wash volumeElution volume2Whole Blood Column3 mL3 mL3x3 mL5 mL
ABCDE1Max volume of whole bloodColumn rinse volumeCell wash volumeElution volume2Whole Blood Column3 mL3 mL3x3 mL5 mL
?. [Example of magnetic separation with medium capacity columns:]
Place the column in a magnetic separator that fits the column.
?. [Example of magnetic separation with medium capacity columns:]
Rinse the column with 3mL of cell separation buffer.
?. [Example of magnetic separation with medium capacity columns:]
Add the labeled whole blood to the column through a 30 µm filter and collect the fraction containing the unlabeled cells.
?. [Example of magnetic separation with medium capacity columns:]
Wash the cells in the column 3 times with 3 mL of buffer and collect the fraction containing the unlabeled cells. Combine with the collected fraction from step 3. These cells may be useful as controls, to monitor purity/yield, or other purposes.
?. [Example of magnetic separation with medium capacity columns:]
Take away the column from the magnet and place it on a tube. Then add 5 mL of elution buffer and flush out the magnetically labeled fraction with a plunger or supplied device. These are the positively isolated cells of interest; do not discard.
TITLE: CD3 Cell Density in Substantia Nigra and Cerebral Peduncle Image Analysis
AUTHORS: Hemanth Ramesh Nelvagal, Toby J Curless, Zane Jaunmuktane

[DESCRIPTION]
QuPath is a bioimage analysis software designed for digital pathology and whole slide image analysis. This protocol describes how to measure CD3 density in the substantia nigra and cerebral peduncle using haematoxylin and DAB-stained brain sections.




[STEPS]
SECTION: Annotation
1. Manually annotate the substantia nigra and cerebral peduncle on NZConnect (Hamamatsu), a web-based whole-slide image (WSI) viewer. 
SECTION: Annotation
2. Download the annotations using a Python script then import into QuPath [1] using a Groovy script. 

SECTION: QuPath Deconvolution and CD3 Density Measurement
3. In QuPath, set the colour deconvolution to facilitate the detection of haematoxylin and DAB staining on CD3 IHC-stained sections.
SECTION: QuPath Deconvolution and CD3 Density Measurement
4. Segment all cell nuclei using StarDist [2] via the QuPath StarDist extension [3];followy with an object classifier to classify CD3-positive cells.

SECTION: QuPath Deconvolution and CD3 Density Measurement
5. Calculate CD3-positive cell density by the number of CD3-positive cells divided by the area of the region of interest (CD3-positive cells per mm^2).
TITLE: Genomic DNA extraction and PCR
AUTHORS: Mark Dewitt & Julia Wong

[STEPS]
?. [Extraction]
Count cells
The amount of cells per µL of QuickExtract is flexible, depending on desired application. For assessment of editing outcomes (e.g. from pools of cells edited with the Cas9 RNP using our other protocols), use sufficient cells to paint in accurate picture from a single PCR (>1000 cells per PCR, ideally, in
?. [Extraction]
Determine volume of cells and volume of QuickExtract DNA extraction solution needed to achieve concentration of 500K cells per 200 µL QE solution (2500 cells per µL)
?. [Extraction]
Spin down appropriate volume of cells: 300xg, 10 minutes
?. [Extraction]
Resuspend in appropriate volume of QE solution, transfer to 1.5mL tubes
?. [Extraction]
Pre-heat a heating block to 65°C
?. [Extraction]
Vortex samples thoroughly for 15 seconds each.
?. [Extraction]
Heat samples on block (65°C) for 6 minutes
?. [Extraction]
Take samples off block, pre-heat block to 98°C (It’s very important for block to reach this temperature! Not enough heat can lead to a failed extraction.)
?. [Extraction]
Vortex samples again, 15 seconds each.
?. [Extraction]
Heat samples on block (98°C) for 2 minutes.
?. [Initial PCR]
Mix for 1rxn   AB1uLcomponent229H2O3105x Q5 Reaction Buffer (NEB)4110mM dNTPs52.510uM Forward Primer62.510uM Reverse Primer70.5Q5 DNA polymerase (NEB 2U/uL)85extract DNArun PCR:1.98C 30s2.98C 10s3.66C 30s4. 72C 20s5. #2-4, 27 cycles6.72C 2min**This final extenstion time may not be necessary for our short amplicons, and might be contributing to unwanted re-annealing and heteroduplexing. The products of this initial PCR often contain heteroduplexes that can interfere with downstream analysis. To get rid of heteroduplexes, we use a "reconditioning" step (see next.)
?. [Reconditioning PCR]
The products of this initial PCR often contain heteroduplexes that can interfere with downstream analysis. To get rid of heteroduplexes, we use a "reconditioning" step (see next.)
?. [Reconditioning PCR]
Reconditioning PCRThis is essentially a 10-fold dilution of the initial PCR products in more master mix.Mix for 1rxn   AB1uLcomponent229H2O3105x Q5 Reaction Buffer (NEB)4110mM dNTPs52.510uM Forward Primer62.510uM Reverse Primer71.0*Q5 DNA polymerase (NEB 2U/uL)85INITIAL PCR PRODUCT *0.5uL is fine too, but we have seen that using double the concentration of polymerase in the reconditioning master mix helps to further eliminate heteroduplexing.run PCR:1.98C 30s2.98C 10s3.66C 30s4. 72C 20s5. #2-4, 27 cyclesno final extension
?. Gel: Add 1-2 µL 6X Purple Loading Dye to 5µL of reconditioned PCR pdt,  run on either 2% agarose or 4-20% TBE acrylamide (to confirm successful elimination of heteroduplexes) at 180V for ~40 minutes.
TITLE: SOLUTION- 06 - Lysis Buffer
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This recipe is used in the following protocols:</div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 01a - </span><span>Isolation of Human PBMC from Buffy Coat</span></div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 01b - </span><span>Isolation of Human PBMC from Whole Blood</span></div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 02 - </span><span>CD4+ T cell Isolation from PBMC with “Dynabeads CD4 Positive Isolation Kit”</span></div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 03 -</span><span> TEFF+TREG Isolation from PBMC with “Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit”</span></div><div class = "text-block"><span style = "font-weight:bold;">- PMN- 01a - </span><span>Isolation of Human PMN from Buffy Coat</span></div><div class = "text-block"><span style = "font-weight:bold;">- PMN- 01b - </span><span>Isolation of Human PMN from Whole Blood</span></div></div>

[STEPS]
TITLE: Microsomal membrane isolation
AUTHORS: Rania Abou El Asrar, Peter Vangheluwe

[DESCRIPTION]
Microsomal membrane isolation 

[STEPS]
SECTION: Harvest cells
1. Harvest cells by detachment with phosphate buffered saline (PBS, Sigma, D8537-500ml) + 0.2% EDTA.

SECTION: Harvest cells
2. Pellet cells by centrifugation (5 min, 400 gavg, 4°C).
SECTION: Harvest cells
3. Wash cell pellet in phosphate buffered saline (PBS, Sigma, D8537-500ml). Repeat 2 times. 
SECTION: Harvest cells
4. Pellet cells by centrifugation (5 min, 400 gavg, 4°C).
SECTION: Lyse cells
5. Resuspend pellet in hypnotic LIS buffer (10 mM Tris HCl pH 7.5, 0.5 mM MgCl2, 1 mM DTT) supplemented with protease inhibitor (Sigmafast, Sigma, S8830-20TAB).
SECTION: Lyse cells
6. Incubate 15 min on ice.
SECTION: Lyse cells
10. Centrifuge lysate (10min, 1000 gavg, 4°C) to pellet and remove nuclear fraction.
SECTION: Isolate microsomal fraction
12. Centrifuge supernatant from previous step (20min, 15 000 gavg, 4°C) to pellet the mitochondrial/lysosomal fraction. 
SECTION: Lyse cells
11. Collect supernatant to continue isolation.
SECTION: Isolate microsomal fraction
13. Collect supernatant to continue isolation (pelleted mitochondrial/lysosomal fraction can be stored for future analysis).
SECTION: Isolate microsomal fraction
14. Centrifuge supernatant from previous step (35 min, 140 000 gavg, 4°C) to pellet the microsomal fraction.
SECTION: Isolate microsomal fraction
15. Resuspend pellets in 0.25 M sucrose, 1 mM DTT and supplemented with protease inhibitors. 
SECTION: Lyse cells
7. Transfer the suspension to a dounce homogenizer and complete 60 up-and-down strokes. 
SECTION: Lyse cells
8. Add an equal volume of 1 M buffer (0.5 M sucrose, 10 mM Tris HCl, pH 7.5, 40 µM CaCl2, 1 mM DTT) supplemented with protease inhibitor.
SECTION: Lyse cells
9. Complete another 30 up-and-down strokes. 
SECTION: Isolate microsomal fraction
16. Snap freeze the resuspended mitochondrial/lysosomal and microsomal fractions in liquid nitrogen. 
SECTION: Isolate microsomal fraction
17. Store at -80 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Reference:&nbsp;<br />JB Waterbury &amp; JM Willey. Isolation and growth of marine planktonic cyanobacteria. 1988. Methods in Enzymology 167: 100-105.&nbsp;

[GUIDELINES]
<strong>Reference:&nbsp;</strong><br />JB Waterbury &amp; JM Willey. Isolation and growth of marine planktonic cyanobacteria. 1988. Methods in Enzymology 167: 100-105.&nbsp;<br /><br /><strong>Formulation:&nbsp;</strong><br />
<table>
<tbody>
<tr>
<td><strong>Stock Solution</strong></td>
<td><strong>Amount</strong></td>
<td><strong>Final Concentration</strong></td>
</tr>
<tr>
<td>EDTA, disodium salt, 1g/L</td>
<td>5.57 ml</td>
<td>15 &micro;M</td>
</tr>
<tr>
<td>K<sub>2</sub>HPO<sub>4</sub>, anhydrous, 6.1g/L</td>
<td>2.57 ml</td>
<td>90&nbsp;&micro;M</td>
</tr>
<tr>
<td>NaNO<sub>3</sub>, 30%</td>
<td>2.57 ml</td>
<td>9 mM</td>
</tr>
<tr>
<td>Na<sub>2</sub>CO<sub>3</sub>&middot;H<sub>2</sub>O, 4g/L</td>
<td>2.65 ml</td>
<td>100&nbsp;&micro;M</td>
</tr>
<tr>
<td>CTMM<sup>1</sup></td>
<td>1.00 ml</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Vit. B12, 10mg/ml</td>
<td>0.10 ml</td>
<td>1 &micro;g/L</td>
</tr>
<tr>
<td>Seawater</td>
<td>750 ml</td>
<td>75%</td>
</tr>
<tr>
<td>DDH<sub>2</sub>O</td>
<td>250 ml</td>
<td>25%</td>
</tr>
</tbody>
</table>
<sup>1</sup> CTMM contains 1.17 &micro;M Na<sub>2</sub>EDTA, 8 nM Zn, 5 nM Co, 90 nM Mn, 3 nM Mo, 10 nM Se, 10 nM Ni and 1.17 &micro;M Fe<br /><br /><strong>Note:</strong><br />Some <em>Synechococcus</em> do not grow well in nitrate containing medium and NH<sub>4</sub><sup>+</sup> can be substituted for NO<sub>3</sub><sup>-</sup> as a source of nitrogen (try using 250-500 &micro;M NH<sub>4</sub>Cl)

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is adapted from Benjy Neymotin's implementation of the SP6 in vitro transcription protocol for the purposes of doing 4tU labeled transcripts.</p>

[GUIDELINES]
<p>Currently, the polyA pSP64 plasmids in Gresham lab are DGP 104,105,106,229. These are the 700,900,1200, and the celegans spike ins.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Endoglycosidase H  digestion of GCase 
AUTHORS: Laura  Smith

[DESCRIPTION]
The N-linked glycoprotein profile of GCase trapped in the ER makes it susceptible to digestion by the glycosidase EndoH. Western blotting of GCase following EndoH digestion will yield an additional lower molecular weight band that corresponds to the GCase that is trapped in the ER. 

[STEPS]
SECTION: Principle
1. To study the subcellular localisation and transport of GCase mutants, the processing of its N-linked glycans was monitored by performing Endo H F digestions. Endo H cleaves only high mannose structures and hybrid structures.

The N-linked glycoprotein profile of GCase trapped in the ER makes it susceptible to digestion by the glycosidase EndoH. Western blotting of GCase following EndoH digestion will yield an additional lower molecular weight band that corresponds to the GCase that is trapped in the ER. 
SECTION: Method
2. Harvest cells (at least 6 well plate for SH/60 mm plate for MEFs, fibroblasts) by trypsinisation. Pellet cells at 200 xg. Wash once in 1 ml PBS. Pellets can be frozen or continue straight on to digestion.
SECTION: Method
3. Lyse cells in 1% (v/v) TX-100 in PBS supplemented with protease inhibitors (1 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml pepstatin A). Lysates need to be at least 2 mg/ml. Therefore lyse 6-well in 100 ml. Incubate on ice, 15 minutes. Pellet debris at full speed in microfuge, 10 min, 4 °C.

NB. Protease inhibitors toxic. Always wear gloves. Weigh out stocks in fume hood. 
SECTION: Method
4. Measure protein concentration with BCA kit.
SECTION: Method
5. EndoH enzyme and buffers is from New England Biolabs (Cat# P0702L). 

Kit contains:
50,000 units EndoH (500 units/ml)
10X Glycoprotein Denaturing Buffer
10X G5 reaction buffer
SECTION: Method
6. For robust detection of GCase after endoH treatment require 10 (minimum) to 20 µg of protein (maximum) per reaction. 

For very low protein concentrations e.g L444P/L444P lines, up to 70 µg protein can be used in order to visualise bands easily by western blot.
SECTION: Method
7. For each sample there are two treatments: (1) without Endo H (2) with Endo H

In small eppendorf mix:
9 µl sample (10-20 mg) protein*
1 µl 10X Glycoprotein Denaturing buffer

* dilute sample accordingly in H20
SECTION: Method
8. Heat sample in block at 100 °C for 10 minutes (make sure tubes are tightly sealed otherwise will pop open and sample evaporates).
SECTION: Method
9. Briefly centrifuge tubes to collect reaction at bottom of tube.
SECTION: Method
10. To each tube containing denatured sample add 10 ml of reaction mixture:

 
            Without EndoH      With EndoH        10X G5 reaction buffer      2 ml      2 ml        EndoH (1000 units)      0 ml      2 ml        H20      8 ml      6 ml   
 For multiple samples it is best to make a master mix.
SECTION: Method
11. Incubate at 37 °C for 1 hour (can be up to 3 hours).
SECTION: Method
12. Stop reaction by adding 5 µl of NuPAGE 4X gel loading buffer and 1 µl 10X reducing agent. Proceed to western blot or freeze samples at -20 °C.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Supplemental notes:</strong> <br /><br />1) Concentration (A<sub>260</sub>/mL) is determined on a UV spectrophotometer (not for iodixanol-purified isolates). </p>
<p>2) Titer (PFU/mL) is determined by plaque assay. </p>
<p>3) 1 A<sub>260</sub> unit of PBCV-1 routinely yields 1.5-2.5 X 10<sup>10</sup> PFU.<br />4) For critical work, a second purification through sucrose gradients or a set of iodixanol gradients may be necessary. <br />5) SAG 3.83 virus purification procedure optimized by Irina Agarkova.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. PiATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment <em>in vivo</em>. We identify a dominant role for hypoxia, marked by HIF1A protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells.</p>

[BEFORE_START]
<p><strong>Prepare before:</strong></p>
<p>2% PFA</p>
<p> </p>
<p>1.25 M Glycin</p>
<p> </p>
<p>single cell suspension solution: 200 U/ml Collagenase in HBSS-FBS (1%))</p>
<p> </p>
<p>Nextera Kit: Illumina #15028212 (containing Tn5, 2X TD buffer)</p>
<p> </p>
<p>Permeabilization buffer: 10 mM Tris ph7.5</p>
<p>                                       10 mM NaCl</p>
<p>                                         3 mM MgCl2</p>
<p>         add fresh 0.1% NP40 (NOTE: NP40 concentration/other detergents can vary from cell type to cell type)</p>
<p> </p>
<p>reverse x-link Buffer: 50 mM Tris-HCl pH 8.0</p>
<p>                                  0.5% Tween20</p>
<p>                                  0.5% Igepal CA-630</p>
<p>                                  5 ng/ml proteinase K</p>
<p> </p>
<p>0.5 M EDTA</p>
<p> </p>
<p>2x NEBNext High Fidelity PCR MasterMix</p>
<p> </p>
<p>Nextera adapters for scATAC (Buenrostro et al, <em>Nature</em> <strong>volume523</strong>, pages486–490 (23 July 2015))</p>
<p> </p>
<p> </p>

[GUIDELINES]
<p><strong>Overview</strong></p>
<p><img src="https://s3.amazonaws.com/pr-journal/z43fw26.png" data-src="https://s3.amazonaws.com/pr-journal/z42fw26.png" data-ofn="Chang_Lab_Figure.png" /></p>

[STEPS]
SECTION: Then make a single cell suspension
?. 
SECTION: Single cell suspension
?. 
SECTION: Single cell suspension
?. 
SECTION: Single cell suspension
?. 
SECTION: Single cell suspension
?. 
SECTION: Single cell suspension
?. 
SECTION: Single cell suspension
?. 
SECTION: Single cell suspension
?. 
SECTION: Fix
?. 
SECTION: Single cell suspension
?. 
SECTION: Fix
?. 
SECTION: Fix
?. 
SECTION: Fix
?. 
SECTION: Fix
?. 
SECTION: Fix
?. 
SECTION: Fix
?. 
SECTION: ATAC
?. 
SECTION: ATAC
?. 
SECTION: ATAC
?. 
SECTION: ATAC
?. 
SECTION: ATAC
?. 
SECTION: ATAC
?. 
SECTION: ATAC
?. 
SECTION: FACS and reverse x-link
?. 
SECTION: FACS and reverse x-link
?. 
SECTION: FACS and reverse x-link
?. 
SECTION: FACS and reverse x-link
?. 
SECTION: FACS and reverse x-link
?. 
SECTION: PCR and	clean up
?. 
SECTION: PCR and	clean up
?. 
SECTION: PCR and	clean up
?. 
SECTION: PCR and	clean up
?. 
SECTION: PCR and	clean up
?. 
SECTION: Preparation
?. 
SECTION: ATAC
?. 
SECTION: Single cell suspension
?. 
?.
TITLE: Mammalian Cell Culture: Freezing
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to freeze cells that are being cultured in a tissue culture flask (T-75 or T-25).</div></div>

[STEPS]
?. [Wash Cells]
Remove media from flask.
?. [Wash Cells]
Using serological pipet, add  DPBS to flask [ for T-25].
4 ml
1 ml
?. [Wash Cells]
Using serological pipet, remove DPBS and dispose into waste beaker.
?. [Wash Cells]
Repeat the above 2 steps, for a total of 2 washes.
?. [Trypsinize]
Add  warmed Trypsin-EDTA to flask [ for T-25].
4 ml
1 ml
?. [Trypsinize]
Wait for cells to detach.
?. [Trypsinize]
Add  warmed cell culture media to flask to neutralize Trypsin-EDTA [ for T-25]
4 ml
1 ml
?. [Pellet Cells]
Using a serological pipet, transfer cell suspension into 15 mL centrifuge tube.
?. [Pellet Cells]
Centrifuge cell suspension at   for  .
?. [Resuspend]
Remove bulk of supernatant with serological pipet.
?. [Resuspend]
Remove remaining supernatant with 1000 μL pipette. For small cell pellets, it is best to leave a little supernatant to avoid disturbing the pellet.
?. [Resuspend]
Add  of either FBS or cell culture media and allow to sit for   to make resuspension easier.
950 µl
Some researchers prefer to use FBS as a freezing medium while others prefer whole media.
?. [Resuspend]
Gently pipette mix cell pellet until resuspended.
?. [Resuspend]
Add  DMSO to cell suspension.
50 µl
5% DMSO solution prevents ice crystals from forming in the liquid, minimizing cell rupture during freezing.
?. [Freeze]
Place cryovial in deep freezer cell vial container.
?. [Freeze]
Allow at least   in deep freezer before transferring cryovial to liquid nitrogen tank.
This prevents too rapidly freezing, which may cause cell rupture.
?. [Document]
Update Lab Frozen Storage Inventory to reflect the new cell vial.