Wash the cells 2x with PBS to get rid of free protein. Treat cells by adding fresh media containing regulator for desired time. Updated on January 28, 2019. Remove the spent media and wash the cell lines monolayer with PBS that is free of Ca2+ and Mg2+. (1) Aspirate off the cell media. 9. Both Tris based and PBS based wash buffers can be used in ELISA protocols. This Cell Preparation Guide describes best practices and general protocols for washing, counting and concentrating cells from both abundant and limited cell suspensions (greater than or less than 100000 total cells, respectively) in preparation for use in 10x Genomics Single Cell Protocols. After harvesting and any stimulation procedures, incubate cells on ice for 20 minutes. For unlabeled, fluorochrome-, or biotin-labeled antibodies: Add 100 l of the Slowly add a volume of sterile PBS equal to the original medium volume, being careful not to dislodge the cells. PBS has many uses because it is isotonic and non-toxic to cells. Remove the vial when a small amount of ice remains. 2. 3. Wash the cells once with ice-cold PBS/BSA/Azide (2mL) Notes on Stimulation Surface staining: 1. Add 1.5 ml pre-warmed trypsin. 1. Scan plate. (See also the protocol in "Current Protocols in Molecular Biology") 1. - Prepare the new dishes and/or six well plates which will be used for the new split. After 5 min add 50 uL cold 2-DG. Incubate for at least 30 min at room temperature or 4C in the dark. Prime system Load cells in chamber Wash cells Recover T cells Results PBMC isolation Centrifuge cells as above, wash 1 time with cold PBS and re-centrifuge. Centrifuge cells 5 min at 300 g, 4C. 4. Aliquot 1 ml cells in a 15 ml polypropylene, V-bottomed tube and add 3 ml cold absolute ethanol. Keep in the dark and at 4C until analysis. 4 Pipette trypsin onto the washed cell monolayer using 1ml per 25cm2 of surface area. Regarding our protocol, low cell yields can be . Wash cells twice with PBS + 1% donkey serum for 10 minutes each wash at room temperature. 4. 5. o Resuspend cells in PBS at appropriate concentration for desired super-loading yield. For intracellular bacteria wash the cells in 1 ml sterile PBS 3 times, aspirate wash and then add 300 g/ml of gentamycin (diluted in . 4. Add 10 mL media to each new dish. The T cell washing and concentration protocol takes approximately 15 min on the CTS Rotea system, based on an input volume of 0.25 L. The workflow is summarized below, and the protocol steps are listed in Table 3. Gently swirl the dish to cover all cells with trypsin 5. Wash the cells by PBS 3 times for 5 min each. 2. Using a clean pipette, separate plasma from red blood cells. 2.Remove PBS w/ Mg 2+ and Ca 2+ and add 1ml of 4% paraformaldehyde (PFA) to fix the . Day 3: Fixing cells and mounting the cover slips. This should take approximately 1 - 2 minutes. 2. 3. Gently wash cells off plate and transfer by pipette to a 15 ml conical tube. After the last wash, try to remove all the PBS. Resuspend cells in an appropriate volume of staining buffer, with care to avoid concentrations that will result in formation of cell aggregates. Do not label with too high CFSE concentration if you want to look at cell division after 1-2 days. Grow ES cells in a 96-well plate to be over-confluent. This serves to block Annexin V-FITC binding sites and thus demonstrates the specificity of Annexin V-FITC staining. Aspirate the PBS, then add 350 l lysis buffer. That way you can just decant or pipet out. Culture cells by adding 500 L of culture media containing approximately 5000 cells to the wells of a cell culture plate containing gelatin-coated coverslips. a.Cells are washed with PBS w/ Mg 2+ and Ca 2+ to minimize cell detachment. The final lysate volume should not exceed 10% of the volume of Trizol used for preparing lysate i.e., in our case the final volume of lysate should be less than 1.1 ml. After the last wash, try to remove all the PBS. Fixation and Permeabilization. Decant the excess trypsin. Centrifuge cells as above, wash 1 time with cold PBS and re-centrifuge. Never pipette medium or wash buffer directly onto cells, always add it gently to the side of the vessel to avoid harming the cells. A general protocol for sample preparation. Re-suspend cells using PBS to achieve a concentration < 1 x 10 6 cells/mL. Determine the cell concentration using the standard method for a hemocytometer or other cell counter. Stain in X-Gal Solution; ~3 mL for 10 cm; stain several hours to overnight at 37C. As completely removing it is impossible, try to get the same final volume in all samples. Wash by adding 200 L/well 1X PBS + 0.1% Tween-20 4 times for 5 minutes each at room temperature with gentle shaking. Distinct labeling of a single HaloTag 7 fusion by a HaloTag Ligand and Anti . Add formaldehyde to obtain a final concentration of 4%. Resuspend cells in 0.5-1 ml 1X PBS. Collect cells by centrifugation and aspirate supernatant. Immunocytochemistry Preparation & Fixation Protocol. Centrifuge the collected supernatant (step 8.5.) Recipe for PBS wash buffer . Wash 105 cells in cold 2% FCS-PBS twice and dilute in 100 (l of cold 1% BSA-PBS. Mix gently and remove the wash solution. Quickly invert the plate over to dump media and remove excess liquid by blotting on paper towels. You may get different results with different reagents, times and concentrations hence the need for protocol optimization. 1. ; When cells have reached the desired density/age, remove the culture media from each well and wash twice with PBS. The protocol has been developed by Proteintech's R&D Team. Just spin-down cells and wash with cold PBS trying to avoid to break them. Pipette trypsin/EDTA onto the washed cell monolayer using approximately 1 ml per 25 cm 2 of surface area. 7. Discard supernatants. 2. Place the cell culture plate on ice and wash the cells with ice-cold PBS for 3 times. Add 10 l of 1 mg/ml PI solution (the final concentration being 10g/ml). Wash cells twice in sterile 1 X PBS solution to remove serum and re-suspend the cells in room temperature PBS at 1-10 x 10 6 cells/mL. Pellet cells 3. If using directly labeled primary antibodies, proceed to step 12. Treat cells by adding fresh media containing regulator for desired time. Stop digestion by adding 8 ml media (DMEm/F12). Keep in the dark and at 4C until analysis. Harvest cells into conical tubes and place them on ice at the end of culture period 2. Cells were fixed in 1% formaldehyde and resuspended in lysis buffer. Add 450 uL KRBH/BSA with or without insulin to wells. PBS can be used as a diluent in methods to dry biomolecules, as water molecules within it will be structured around the substance (protein, for example) Repeat twice. Thanks in advance for answering this. Note: Alternatively, rinse cells twice with PBS, incubate in PBS for 30 min., then rinse with PBS. The recommended cell washing and resuspension solution is 1X PBS (calcium and magnesium free) containing 0.04% weight/volume BSA (400 g/ml). Suspend cells in ice-cold PBS/BSA/Azide (50l for each test = 1-2 x . Feed cells 2-3 hours before trypsinization 2 Aspirate media and wash cells with PBS 3 Add trypsin: 96-well plate - 30 ul/well 24-well plate - 100 ul/well 6-well plate - 500 ul/well 10 cm dish - 2 ml/plate 4 Incubate for 5 min @37C 5. 5. Remove growth medium from the cells by decanting or aspirating the medium. A cell pellet may not be visible after the fixation step because fixed cells aggregate less well and therefore tend to spread out at the bottom of the tube. Use a minimum of 1x10 6 cells per tube. Wash cells once with 10 mL (per 10 cm dish) PBS -/- then aspirate the PBS. Remove remaining solution and air dry overnight at room temperature. PBS + 2% FBS; PBS + 0.1% BSA) Wash cells twice and resuspend at 1-2 x 10 6 cells/ml. Re-suspend cell pellet in 0.25 ml of PBS, add 5 l of 10 mg/ml Rnase A (the final concentration being 0.2-0.5 mg/ml). 6. ; Add 300-400 L of 2-4% Formaldehyde Fixative Solution to each well . Arrange a single-cell suspension of cells of interest. Transfer 100 l of the solution (~1 x 10 5 cells) to a 5 ml culture tube. Protocol for Immunostaining of cells Reagents: - media without serum - PBS++ (containing 0.9 mMCaCl2, 0.52 mMMgCl2 and 0.16 mM MgSO4) . Wash cells 2 X in Tris / PBS. Fix the cells with 2-4% paraformaldehyde (PFA) for 10-20 min at RT or with cold methanol, acetone (1-10 min) at -20C. 6. Rinse cells briefly in PBS. Add in cold RIPA lysis buffer (1ml for 10 7 cells). 4. Wash cells twice with 1x phosphate buffered saline (PBS) (Cat. In general, pre-coated plates require fewer wash steps than DIY . Prepare insulin in KRBH/BSA (0.6uL insulin/mL), needing 0.5 mL per well) Wash cells 2x with warm PBS -/-. 3 Wash the cell monolayer with 1-2 ml of PBS without Ca2+/Mg2+ (CMF-PBS). 1. 3. Primary cells, stem cells and other sensitive cell types may require washing and suspension in alternative buffers to maximize viability. Gently vortex and incubate at room . Resuspend the cells in 200ml of FACS buffer and filter samples prior to running. Remove fixative and gently wash cells 2 times with 2 ml PBS. Transfer to 1.5 mL eppendorf tube. o Filter cells through 40 m strainers (e.g. Label 10E6 cellule/ml in 5 M CFSE final (conc stock 1000x ). After washing off the surface stain use the eBioscience Foxp3 kit for fixing and staining. Wash with 10 ml pre-warmed PBS Add PBS to the side of the dish, and slowly tilt dish to gently wash the cells. 4. 3. WASHING RED BLOOD CELLS 1. Seed cells. Harvest, wash the cells (single cell suspension) and adjust cell number to a concentration of 1-5106 cells/ml in ice cold FACS Buffer (PBS, 0.5-1% BSA or 5-10% FBS, 0.1% NaN3 sodium azide*). Prepare lysis buffer with detergent (10 mL) and without detergent (50 mL) Wash cells 2x with ice cold PBS (10 mL for a 10cm dish, 25 mL for a 15cm dish) Add 1 mL lysis buffer and scrape cells with cell scraper. Before starting the protocol: - Bring cell media, PBS, and trypsin to room temperature. -xtrios- We wash cells with cold PBS before extracting protein. with PBS. (~ 1,500 cells/l or higher, use cost per cell calculator). 2. While cells are incubating, remove medium from fresh feeder plates and add fresh, pre-warmed medium 7. Wash 1-3 times as described throughout this protocol. Wash 3 X in PBS / Tris (2nd wash 10 min). Incubate end over end for 15-30 min at 4C to lyse. Phosphate buffered saline (abbreviated as PBS) is a buffer solution commonly used in biological research. For longer time points, you can increase the CFSE concentration. If losing the cells in this wash step is a real. Add 1 L of Fixable Viability Dye per 1 mL of cells and vortex immediately. At the time of fixation, cells should be ~70-80% confluent in single layer. 4. Figure: In-cell western data showing detection of tubulin in permeabilized Capan-2 cells. Harvest cells in 50 ml conical tubes, quickly wash cells with PBS once by spinning at 500. g . *Do not add sodium azide to buffers if you are concerned with recovering cell [] Wash the cells in two to four volumes of PBS centrifuging at 300g for 5 minutes. P3813). Wash cells twice in Flow Cytometry Staining Buffer or equivalent. 3. . Protocol. If a final concentration of 5 uM is desired, add .5 uL of the 10 mM reconstituted solution . Digest for 5 minutes at 37C. Wash the cells in stain buffer (with BSA or FBS) and resuspend in a volume that is appropriate for cell counting. 5. Apply collagen coating solution. . Adjust Mouse Endothelial Cell suspension to a concentration of at least 0.5 x 106 cells/ml in 1-2% BSA in 1X PBS with Calcium & Magnesium. Keep the cells in the dark on ice or at 4C in a fridge until your scheduled time for analysis. Discard supernatant in appropriate waste container. Permeabilization If the target protein is intracellular, it is very important to permeabilize the cells. (Avoid any protein or serum at this step as it will hinder the hydrogel polymerization process). Incubate overnight at 4-8C. Wash by centrifugation with excess 1X PBS. Prepare collagen solution 10ug/ml in 10% IPA, 0.1M acetic acid. Harvest, wash the cells (single cell suspension) and adjust cell number to a concentration of 1-5106 cells/ml in ice cold FACS Buffer (PBS, 0.5-1% BSA or 5-10% FBS, 0.1% NaN3 sodium azide*). Wash the cell pellet with PBS by centrifuging at 200 x g for 5 minutes. It is a salty solution containing sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium phosphate. Add 2 ml 1X Trypsin/EDTA. Keep cells on ice until fixation. (3) Aspirate off the PBS. The procedure follows. 4. Lyse cells by adding 1X SDS sample buffer (100 l per well of 6-well plate or 500 l for a 10 cm diameter plate). 3. If necessary, stain for surface markers as per usual FACS protocol. Question: What buffers can be used for washing and cell resuspension? Wash the cells 3 times by centrifugation at 1500 rpm for 5 minutes and resuspend them in 200 l to 1ml of ice cold FACS buffer*. But we were to wash cells before trypsinising, we use warm PBS. Briefly, the subculturing protocol for adherent cells is as follows: media is removed and cells are washed once with PBS. Try to remove the culture medium as much as possible from the dish. Cells can be left in PBS for longer times without negatively affecting staining. Aspirate medium, then briefly wash cells seeded on clean glass coverslips with 1X PBS. Sigma-Aldrich recommended collagen coating protocol Protect from light. When resuspending a pellet or triturating to mix cells, do so gently. Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of ~1x 10 6 cells/ml. Wash in PBS or culture medium. BSA is added to minimize cell losses and aggregation. Add 9 mL media to trypsinized cells. Rotate flask to cover the monolayer with trypsin. Add 10 l of 1 mg/ml PI solution (the final concentration being 10g/ml). This protocol allowed us to study the functional effects of a TNT mediated process of an organelle transfer between MDMs both in vitro and using the same staining protocol, mouse alveolar macrophages in vivo . The distortion of the cell morphology is something to bear in mind when interpreting the images. Add 1 mL trypsin and allow to sit in the hood for 2-5 min. Aspirate media from cultures; wash cells with 1X PBS; aspirate. First, you have to get rid of red cells. In a 384- Imaging well-plate (Cat. Carefully wash cultured cells with pre-chilled PBS for 2 times. Resuspend cells in 0.5-1 ml 1X PBS. Wash the pellet with PBS. Starve cells >3h in 0.5% FBS. University of Kansas Washing cell pellets generally mean you have to re-suspend the cells with the washing agent (usually PBS) and then re-centrifuge it. 4. Intracellular staining of Tbet, Bcl6, Foxp3, KI-67, Bcl2, etc. Lyse cells by adding 1X SDS sample buffer (100 l per well of 6-well plate or 500 l for a 10 cm diameter plate). If the cells are known to attach aggressively, repeat the wash step. Fix in 4% paraformaldehyde in PBS; 15 minutes at Room Temp; 5 mL for 10 cm, 2 mL for 6 cm. Mitra, S.C. De Rosa, N. Watanabe Cell Preparation 1. o Merge cells from all tubes at equal ratios in 1 ml Staining buffer, spin 5 minutes 350g at 4C. Keep cells in blocking buffer for 30 min at RT. Centrifuge whole blood sample. Incubate on ice for 30-60 minutes in the dark. Immunofluorescence Protocol (for adherent cells) info@arigobio.com www.arigobio.com 3 / 4 Process: Fixation: Aspirate culture medium and rinse the cells with PBS twice. No. Chromatin was sheared to 100- . Briefly, disrupt the cell pellet by "racking" the tube, resuspend the cells in erythrocyte lysis solution, vortex the cell suspension, incubate the cell suspension for 10 minutes at room temperature, and wash the cells with Wash Buffer from the lysing kit. (eg. Wipe the outside of the vial of cells with 70% ethanol or isopropanol. Depending on your postion in the protocol 1-3-5 wash steps maybe required. for 5 min at 4 C and resupension. 8. Protocol. 4. Protocol. Wash cells 2 X in Tris / PBS. Transfer to a microscope, and capture images. Remove the coverslips from the antigen retrieval buffer and immerse them, with the side containing the cells facing up, in PBS, in the 6-well tissue culture plates. 7. This step will require optimization. After the last wash, gently pipette out any residual liquid and blot plate dry. But we usually, levae the plate with cells on ice bucket for a few min. to cool the cells down to reduce cell shock and to reduce activity of proteases and other enzymes. (If using PRBCs, start at Step 3.) Wash quickly three times with PBS and let dry before plating cells. Wait 30 min. Aspirate the plate media. Rotate . First step: Wash cells with PBS If working with suspension cells, this step is very easy. Figure 9. Protocol. Incubate for 30 minutes at 2-8C. Use a minimum of 1x10 6 cells per tube If necessary, stain for surface markers as per usual FACS protocol Wash cells with PBS Suspend cells in 0.5mL PBS and 0.5mL fixation buffer. Repeat this wash step if the cells are known to adhere strongly. Fix cells ~20 - 30 hours later. Grow cells to confluency on p150 plate. No. Immunocytochemistry General Protocol. Typically do triplicate measurements. Add 1 ml PBS. A 96-well tissue culture plate was seeded . CLS4851), add 40 L of cell suspension per well. The purpose of this step is to block non-specific . It is used to rinse containers containing cells. For example, put the required volumes of cell media into the new dishes. ChIP-seq: Cells were grown according to the approved ENCODE cell culture protocols. Aspirate media from cultures; wash cells with 1X PBS; aspirate. Wash cells twice in perm buffer and once in FACS buffer. This protocol is optimized for porcine hearts but can be adapted for use with other large animals. Put them on a low-speed rotating shaker for 15 min at 4C. . -scolix- Add 1 mL of 0.1 mg/mL Poly-D-lysine solution into a well for 15 minutes at room temperature. Enter the email address you signed up with and we'll email you a reset link. Flow cytometry (FACS) staining protocol (Cell surface staining) 1. Wash cells in PBS three times for 5 min. A general protocol for sample preparation. To get a single cell solution, which is necessary for both splitting and freezing, you will probably always need the PBS wash step, sometimes twice. Be sure to keep all samples on ice. Put the plate into a humidified chamber Warm PBS and Media in water bath. CFSE Staining Protocol. Sample prep, SDS-PAGE and transfer. Heart is washed by flushing with PBS, whereafter endothelial cells are detached by collagenase incubation and the cells can then be collected immediately after the incubation and plated within an hour after the heart is isolated from a pig. 2. Suspend cells in 0.5mL PBS and 0.5mL fixation buffer. The 89 Zr-oxine cell radiolabeling procedure consists of three main steps: (1) Preparation of the cells in an labeling (incubation) buffer which can be either PBS or a similar non-protein . Sample Preparation: Grow cultured cells on cover slips or in wells overnight at 37C. Follow the protocol for surface staining. If working with adherent (that is attached) cells, wash cells with cold PBS (to prevent cells detaching, use PBS containing calcium and magnesium) and make sure to remove all the PBS from the cells before . 2. Discard supernatant, making sure not to disturb the RBC pellet. Wash cells with PBS. Add 0.1-10 g/ml of the primary antibody (UNCONJUGATED). Fix for 15 min at room temperature. in PBS-BSA (keep cells dark) - wash 3x with PBS++ - dip coverslip in demi water for a few seconds - mount cells up site down in mounting solution, let dry for 30' in the dark . Quickly thaw cells in a 37C water bath by gently swirling the vial. Wash the cells with sterile PBS to remove residual medium. Live-Cell Labeling (No-Wash Protocol) . Add approximately 1x10e6 cells to each flow cytometry tube and wash with 1 ml of 0.1% saponin or Tween 20 diluted in PBS with 2% FBS added. Proceed to running samples on the flow cytometer. Animal Cell Culture Protocol Procedure 1 Eyeball the cells - View cultures using an inverted microscope to assess the degree of confluency and . Flow cytometry (FACS) staining protocol (Cell surface staining) 1. Check cells for trypsinization, and if necessary tap the cells. Add CFSE solution to the chosen final concentration. It can be used to dilute substances. Flowmi cell strainer). Answer: To prepare single cells for Chromium Single Cell applications, it is recommended to use 1X PBS (calcium and magnesium-free) containing 0.04% weight/volume BSA (400 g/ml) for washing and resuspension.Fresh and frozen/thawed PBMC samples and cell lines have been tested with this buffer. Protocol for staining whole cells with PI for cell cycle analysis: Method: Harvest cells and prepare single cell suspension in buffer (e.g. Spin cells at 1000- 12000 rpm at 4C or room temperature for 5 minutes. Protocol for Immunostaining of cells Reagents: - media without serum - PBS++ (containing 0.9 mMCaCl2, 0.52 mMMgCl2 and 0.16 mM MgSO4) . Wash 3 X in PBS / Tris (2nd wash 10 min). Scrap cells off to clean 1.5ml eppendorf tubes with a clean, cold scraper. It may, however, require further adaptation for each specific experiment. Cells are then incubated in the ELISA plate for up to 3 hours, this can depend on application. Store cells in PBS + 0.1% sodium azide. Keep cells on ice until fixation. In the event that there are few cells available, aliquot about 100 (l of cold 1% BSA . 1. In a biosafety cabinet, twist the cap a quarter-turn to relieve internal pressure and then retighten. at 300 x g, 5 min, 4 C. Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml collection tube, and centrifuge for 2 min at full speed. Re-suspend cell pellet in 0.25 ml of PBS, add 5 l of 10 mg/ml Rnase A (the final concentration being 0.2-0.5 mg/ml). protein A/G-agarose beads Specific antibody (MAb or PAb) Protocol: DAY 1 1. through a 70-m cell strainer. in PBS-BSA (keep cells dark) - wash 3x with PBS++ - dip coverslip in demi water for a few seconds - mount cells up site down in mounting solution, let dry for 30' in the dark . 2. Add 50 ul of Bradley Lysis Buffer containing proteinase K. Replace lid and seal the plate with parafilm. Incubate at 37C for 1 hour. Resuspend cells in 1 ml of 0.1% saponin (or Tween 20) + 2% FBS and incubate for 30 minutes at RT. Procedure for Lysis of Adherent Cells 1. Place slides and filters into appropriate slots in the cytospin with the cardboard filters facing the center of the cytospin. Immediately scrape the cells off the plate and . Gently vortex and incubate at room temperature for 20 minutes Heat the coverslips at 95C for 10 min. After centrifugation at 300 x g, 5 min, 4 C re-suspend the cells in Stellate Cell/Endothelial Cell separation medium and perform a cell count for all remaining cells as described in step 4.6. . in T cells. To red cells in tube, add PBS or saline, filling tube almost 3/4 full. Incubate at 37o for 15 minutes 6. Count cells using a hemocytometer or alternative method. Remove supernatant by aspiration or rapid decanting. Stain in X-Gal Solution; ~3 mL for 10 cm; stain several hours to overnight at 37C. Wash cells twice in azide-free and serum/protein-free PBS. 1.Remove media and briefly wash cells (cell can be grown on chamber slides or on 24, 12, 6- well plates) with PBS w/ Mg 2+ and Ca 2+. In other words, it's isotonic to human solutions, so it's less likely to cause cell damage, toxicity, or unwanted precipitation in biological, medical . Wash cells in PBS-CMF 2X. (See also the protocol in "Current Protocols in Molecular Biology") 1. Rinse cells twice with PBS, then wash 3 x 5 min. PBS or phosphate-buffered saline is a buffer solution that is particularly valuable because it mimic the ion concentration, osmolarity, and pH of human body fluids. Centrifuge for approximately 60 seconds. Wash the cell monolayer with PBS without Ca 2+ /Mg 2+. Washing cell monolayer with ice-cold PBS is not recommended as washing can contribute to mRNA degradation. Incubate at 37C for 1 hour. Cytospin Protocol. Fixing and permeabilizing your cells affects the cell morphology and the availability of the antigen you are trying to detect. Wash the cells 1 time with blocking buffer (1% BSA in 1X PBS with Calcium & Magnesium). NUNC recommended collagen coating protocol. The buffer helps to maintain a constant pH. Remove the growth medium and add 2 ml PBS (37C). Protocol I. Fix the cells for 10-20 min. *Do not add sodium azide to buffers if you are concerned with recovering cell [] Remove the PBS and add 2 ml of 4% paraformaldehyde/PBS; incubate 20 minutes at 4 C (place dish in refrigerator) 3. Protocol prepared by D.K. Rinse coverslip with 1X PBS 3 times for 3 min each. Cell Culture Protocol 5: Subculture of Suspension Cell Lines (sigmaaldrich.com) Agriculture; Animal Tissue Culture; Biochemical Analysis; Biochemistry; Replace wash solution with 1X PBS. No 556570). Resuspend cells at 1-10 x 10 6 cells/mL in azide-free and serum/protein-free PBS.