1. Grow cells under the desired conditions to 0.5-1x10⁷ cells/mL. Collect up to 1-2x10⁷ cells per sample. All the following steps can be performed in a single 1.7mL microfuge tube per sample.
2. Pellet the cells (a few seconds in a microfuge). Resuspend the cells in 300μl H₂O. Be sure the pellets have been dispersed before the next step.
3. Add 0.7mL 100% ethanol and vortex; incubate at 4°C for at least 30 minutes.
4. Pellet the cells as before and resuspend in 1mL of 50mM sodium citrate, pH 7.4.
5. Sonicate the cells (10 pulses of 0.9 seconds each, setting of –32 to –40).
6. Pellet the cells again and resuspend them in 1mL Na-citrate containing 0.25mg/mL RNase A. Incubate at 50-55°C for 1 hour.
7. Add 25μL of 20mg/mL Proteinase K and continue the incubation for an additional hour.
8. Pellet the cells again and resuspend the pellets in 500μl of 50mM Na-citrate. [But go instead to “Alternative” in Step 10 if your cell concentration is not very high.]
9. Prepare a batch of 1x Sytox Green (= 1μM) in 50mM Na-citrate. The stock solution provided by the vendor is 5mM = 5000x, in DMSO. Note: the manufacturer recommends wearing two pairs of gloves when handling the stock solution. The Sytox stock solution takes a while to thaw, so plan ahead.
10. Pipette out 1 ml aliquots of the 1x Sytox solution into FACS analysis tubes (Falcon 2054). Add 50μL of the cell sample (or enough to get ~1-2x10⁶cells; the goal is to have 100-200 events per second within the gated region when the samples are being read). Incubate 1 hour to overnight at 4°C; stained samples can be stored at 4°C for several weeks. [Alternative: If your sample is low-concentration, you can simply add 1 ml of 2x Sytox to the cell sample immediately after step 7, and you’ll be good to go.]
11. Use FL1 on the FACSCAN instrument. Sytox Green fluoresces more strongly than propidium iodide, so the voltage/gain won’t have to be set as high as with propidium iodide.

Sytox Green: Invitrogen, catalog # S7020

RNase A Stock Solution (10mg/mL)

• 0.2g Ribonuclease A (Sigma #R-5503)
• 200µL 1M Tris-HCl, pH 7.5
• 60µL 5M NaCl
• Bring volume up to 20mL with H₂O
• Boil 15 minutes.
• Let cool slowly to room temperature.
• Aliquot and store at -20°C.

Proteinase K Stock Solution (20mg/mL)

• Stock solution is 20mg/mL in
• 10mM Tris pH 8.0
• 1mM CaCl₂
• 50% glycerol
• To make 5mL of stock solution, dissolve 100mg Proteinase K in:
• 3.125mL 80% glycerol*
• 50μl 1 M Tris pH 8.0
• 50μl 100mM CaCl₂
• 1.775mL H₂O
• = 5mL total; dispense in 1mL portions and store at –20°C.
• *autoclaved stock; easier to deal with than 100% glycerol that’s not been sterilized

Reagents:

 This protocol has been found useful for labeling both primary cells and cell lines with the fluorescent probe CFDA-SA (carboxyfluorescein diacetate succinimidyl ester). This probe is often referred to incorrectly in the literature as "CFSE" - it should not be confused with carboxyfluorescein succinimidyl ester (the real CFSE), which is not the diacetate form and is not cell-permeable. The correct reagent can be obtained from Molecular Probes, catalog number C-1157.

Stock and Storage:

 We prepare CFDA-SE at a stock concentration 1000-fold higher than the final usage concentration (for example, 2mM if the final concentration is 2µM) in dry DMSO. Aliquot into single-usage vials and store over desiccant at -20ºC. CFDA-SE will hydrolyze quickly at room temperature in the presence of water, and much more slowly at -20ºC under desiccating conditions. Aliquoted stocks should be used for no more than 2 months. If your cells show decreased labeling with the same stock of CFDA-SE, hydrolysis is likely the cause.

Labeling Concentration and Conditions:

 Cells are usually labeled at a final CFDA-SE concentration of 0.5-5µM. The literature reports concentrations ranging from 0.2-10µM and even higher. For best results, do a titration and find the lowest concentration of CFDA-SE that will give effective cell labeling - this will vary from cell type to cell type, and also with the application. CFDA-SE labeling is somewhat toxic and can induce growth arrest and apoptosis in some cell types - therefore, it is important to find the lowest acceptable labeling concentration and check the viability after labeling. As a rough guide, 0.5-2µM is usually enough for in vitro experiments - cell tracking and generational analysis in transplanted cells may require 2-5µM. Incubation time is usually from 5-10 minutes - again, titrate to find the minimal effective conditions. We usually label in PBS or HBSS containing 0.1% BSA. All post-labeling washes should be carried out in complete media (such as RPMI with 10% FBS) - your intended tissue culture media is ideal. The high protein concentration inactivates unreacted CFDA-SE.

1. Suspend your cells in PBS or HBSS containing 0.1% BSA. Cell concentration can range widely from 1x10⁶ cell/mL (for in vitro experiments) up to 5x10⁷ cells/mL (for adoptive transfer). The cells should be in single cell suspension - if necessary, filter them through nylon mesh immediately prior to labeling. Total reaction volumes should not exceed 4mL in a 15mL tube, so prepare cell suspensions at no greater than 2mL each.
2. Prepare a solution of CFDA-SE from your DMSO stock in PBS/0.1% BSA at 2x the final labeling concentration. For example, if you are labeling at 5µM, prepare a 10µM solution. Prepare a volume of CFDA-SE equal to your cell volume above (no more than 2mL per labeling reaction)
3. Add an equal volume of CFDA-SE solution to your cell suspension. Mix gently and incubate for 5-10 minutes at 37ºC
4. Immediately fill the labeling tube to the top with the tissue culture media intended for culture (such as RPMI/10% FBS) and centrifuge. Wash the cells three times with tissue culture media at room temperature. To reduce the amount of unbound CFDA-SE in cells, we usually incubate the cells at 37ºC for 5 minutes after the second wash and prior to the third. This allows free unreacted CFDA-SE to diffuse out of the cells and be removed in the final wash.

This protocol was prepared by the Telford Lab for the NCI Medicine Branch and its friends. 11-18-00

Solutions:

Staining Buffer:

• PBS, pH 7.4-7.6
• 2% heat inactivated BCS
• 0.2% sodium azide

Fixation Buffer (4% paraformaldehyde)

• 14mL 10X PBS
• 10.8mL 37.5% formaldehyde
• 75.2mL dH₂O

Perm Buffer

• Staining Buffer + 0.5% saponin

Superperm Buffer

• 3 parts Perm Buffer + 1 part BCS (filtered)

Staining Protocol

After harvesting and any stimulation procedures, incubate cells on ice for 20 minutes. Keep cells on ice until fixation.

1. Use a minimum of 1x10⁶ cells per tube
2. If necessary, stain for surface markers as per usual FACS protocol
3. Wash cells with PBS
4. Suspend cells in 0.5mL PBS and 0.5mL fixation buffer. Gently vortex and incubate at room temperature for 20 minutes
5. Optional - To store cells after fixation, spin cells down and repeat fixation step, suspend cells in Staining Buffer and store refrigerated for up to 3 days.
6. Wash with PBS

All Intracellular Staining steps should be in saponin containing buffers

1. Wash with Perm Buffer once and then wash once with Superperm Buffer. After fixation, cells can be vortexed in the usual manner
2. Add primary antibody for intracellular staining. It is recommended to use 2x the normal amount that would be used for surface staining, but you may need to optimize by tritration
3. Incubate in the dark at room temperature for 30 minutes
4. Wash with Perm Buffer twice
5. If necessary, add secondary antibody. It is recommended to use 2x the normal amount that would be used for surface staining, but you may need to optimize by tritration. Secondary PE-conjugated reagents are recommended to get the maximum signal.
6. Incubate at room temperature for 30 minutes
7. Wash with Perm Buffer twice
8. Wash with PBS once
9. Wash with Staining Buffer once
10. Resuspend in 300-500mL Staining Buffer and analyze by FACS

Notes:

• commerical fixative and permiabilization solutions are available if preferred
• Depending on cytokine localization, stronger permeabilizing detergents such as Triton-X or NP-40 may be appropriate (0.1% - 1%)

Adapted From:

1. Shimizu Lab/Author Nadine Ottoson http://www.tc.umn.edu/~shimi002/sopicfacs.pdf
2. Think Peptides - Intracellular Cytokine Staining http://www.thinkpeptides.com/PR02TP.pdf
3. Abcam - Intracellular Staining http://www.abcam.com/ps/pdf/protocols/flow_intracellular_staining.pdf

1. Collect cells from culture or tissue. Make a single cell suspension of ~1x10⁶ cells. Wash cells by adding 3mL PBS, vortex gently and centrifuge at 200g for 5 minutes at 4ºC. Resuspend cells in Falcon tube in 50µL PBS
2. Dilute the monoclonals according to results from a previous titration
3. Add 5µL diluted monoclonal antibody to each tube. Shake tubes gently by hand. Place in an ice bath for 15 minutes in the dark
4. Add 3mL PBS to each tube. Spin at 200g for 5 minutes at 4ºC
5. Decant supernatant. Add 400µL 2% paraformaldehyde (pH 7.2-7.4) while vortexing
6. Cap tubes and store at 4ºC wrapped in foil until ready to run
    

References

Jackson, A.L., Warner Preparation, Staining and Analysis by Flow Cytometry of Peripheral Blood Leukocytes. In Manual of Clinical Laboratory Immunology. N.R. Rose, H. Friedman and H.L. Fahey, editors. American Society for Microbiology, Washington D.C., 1986, pp 226-235.

For adherent cells in 100mm plate

1. Transfer media to 50mL centrifuge tube
2. Wash plate with cold PBS and transfer to 50mL tube from above
3. Trypsinize cells and deactivate trypsin with media containing serum and transfer 50mL centrifuge tube from above
4. Fill 50mL centrifuge tube with cold PBS
5. Spin at 1500rpm for 10 minutes
6. There should be a pellet
7. Remove media and resuspend by slowly adding 1mL ice-cold 70% ethanol while vortexing
8. Store at -20ºC overnight, not more than 1 week

When ready to analyze:

1. Spin cells fixed in ethanol at 2000rpm for 15 minutes
2. Remove ethanol
3. Resuspend cells in 0.5mL cold PBS for small pellet and 1mL cold PBS for large pellet and transfer to flow cytometer friendly tube
4. Add 1/20 volume of 10mg/mL RNAse A (in TE buffer)
5. Add 1/40 volume of 1.6mg/mL propidium iodide (in ddH₂O)
6. Incubate at 37ºC for 30 minutes covered
7. Put samples on ice, covered
8. Analyze using flow cytometry within an hour.

Protocol adapted by: T. Landowski, Ph.D., and Alan Pourpak

Binding Buffer:

• 10mM HEPES/NaOH, pH 7.4
• 150mM NaCl
• 5mM KCl
• 1mM MgCl₂
• 1.8mM CaCl₂

PI Solution:

• Propidium iodide 100µg/ml in Binding Buffer

1. 1Wash 2x10⁶ cells with PBS.
2. Dilute FITC-Annexin V to 1µg/ml in binding buffer and resuspend cells in 1ml of this solution (prepared fresh each time)
3. Incubate 10 min. in the dark at room temperature
4. Add 10µl of PI solution for a final concentration of 1µg/ml prior to analysis

1. Wash cells in PBS and resuspend 2 x 10⁶ cells in 0.5 ml PBS.
2. Fix cells by addition of 5 ml of 1% paraformaldehyde. Incubate on ice for 15 min.
3. Wash cells twice and resuspend in 0.5 ml PBS in polypropylene tubes.
4. Permeabilize cells by addition of 3 ml of ice-cold ethanol (70%) for at least 4 hr on ice.
   (Cells may be stored for several weeks in 70% ethanol at -20^oC)
5. Centrifuge sample, wash in PBS and resuspend cells in 50 µl of TdT reaction solution:
   • 10 µl TdT reaction buffer
   • 2.0 µl BrdUTP stock solution
   • 15 U TdT, 5µl CoCL2 (10 mM)
   • 33.5 µl H₂0
6. Incubate cells for 40 min at 37^oC with occasional mixing (or room temp. overnight).
7. Add 1.5 ml rinsing buffer and centrifuge.
8. Add anti-BrdU-FITC antibody (approx. 100 µl) and incubate at room temp. for 1 hr.
9. Add 1ml of PI staining solution, incubate for at least 30 min at room temp. in the dark.
10. Analyze for FITC/PI fluorescence with appropriate controls for instrument setup.

The exact incubation times for fixation, permeabilization, TdT may vary according to cell type. It may be necessary to optimize these for specific cells and applications

The exact incubation times for fixation, permeabilization, TdT may vary according to cell type. It may be necessary to optimize these for specific cells and applications.

Reagents:

TdT Reaction Buffer (5X):

• 1M potassium or sodium cacodylate, 125mM TRIS-HCl, pH 6.6, 1.25mg/ml BSA. Store at -20ºC.

Anti-BrdU-FITC Antibody Solution:

• 0.3µg anti-BrdU-FITC antibody, 0.3% w/v Triton X-100, 1% BSA, PBS to 100µl volume per test.

PI Staining solution:

• Propidium Iodide 5µg/m PI, RNase A (DNase free) 200µg/ml, PBS, pH 7.2-7.4

Many vendors sell kits with pre-prepared buffers and reagents. Alternatively, individual reagents may be purchased separately. The above protocol is a general guide and individual kits may vary in the amounts of reagents used.

1. Add 1µl of 1 mg/ml 7-AAD (1µg/ml final) to approximately 1x10⁶ washed cells suspended in 1ml PBS.
2. Incubate ≥ 30 minutes in the dark on ice.
3. Analyze on flow cytometer with excitation at 488nm. Collect fluorescence emission with a 650nm long-pass or a 670±20nm band-pass filter.

7-AAD is easily excitable at 488nm. The fluorescence emission of the dye has a peak at ~670nm.

From Current Protocols in Flow Cytometry

We would appreciate it if the Cytometry Core Facility is mentioned in the acknowledgement section of any publication which includes results of data produced from the facility, and that an electronic copy of the paper be provided to the facility. This will help us justify upgrading services and equipment on grant applications.

Please cite the facility as: "UACC/ARL Cytometry Core Facility" and include the Cancer Center Support Grant (CCSG - CA 023074)

Methods Section of the Paper

Please use the links below to create the configuration specific to your application. Following is an example of a typical description. Please contact the lab personnel if you wish assistance with the wording:

Two-color flow cytometric analysis was performed using a FACScan flow cytometer (BDBiosciences, San Jose, CA) equipped with an air-cooled 15mW argon ion laser tuned to 488nm. The emission fluorescence of (yada, yada conjugated to IgG...blah blah...) was detected and recorded through a 530/30 bandpass filter in the FL1 channel. (Something red) was detected in the FL2 channel through a 585/42 bandpass filter. List mode data files consisting of 10,000 events gated on FSC (forward scatter) vs SSC (side scatter) were acquired and analyzed using CellQuest PRO software (BDBiosciences, San Jose, CA) at a rate of 200-400 events per second. Appropriate electronic compensation was adjusted by acquiring the cell populations stained with each dye/fluorophore individually, as well as an unstained control.

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