Monochlorobimane FACS-Staining Protocol

Monochlorobimane FACS-Staining Protocol

Last Updated 11/19/99

Contacts: Steve De Rosa (derosa@fhcrc.org)

Materials	Qty	Location	Order Info
MCB	25mg	Freezer 5 (B-011)	Molecular Probes M-1381
NCS	15-30mL	Freezer 3 (B-010)
Premixed antibody stains*		Fridge 5 (B-011)
Compensation antibodies		Fridge 5 (B-011)
hRPMI (for bimane media)		Cold Room (B-008)	In-lab stock. See Bimane Media (hRPMI) Formulation Protocol for details
defRPMI		Cold Room (B-008)	In-lab stock. See Herzenberg Lab Media Preparation for details
10% Sodium azide
Probenecid		Chemicals G Shelf, B-010	p-[Dipropylsulfamoyl]benzoic acid, Sigma P-8761
Paraformaldehyde (4%)
TruCount Tubes	2 x n	B-011	Becton Dickinson 340334
FACS Lysing Solution		B-011	Becton Dickinson 349202
Conical tubes			15mL conical tubes
96-well plates			Round bottom wells
FACS tubes			Falcon 35-2052 (no caps), 2058 (caps)
Large ice buckets	2

* This protocol works with 1 to 11 color experiments

Protocol prepared May 12, 1998 by Mario Roederer, Michael Anderson, Steve DeRosa, and Jim Andrus

Introduction

Samples, in general, will arrive in the laboratory in the morning or early afternoon. To facilitate a smooth experiment, immediately prior to the time of arrival, the standards should be prepared in the laboratory. Standards should not be prepared too far in advance of bimane staining as this may affect results. Preparation of the standard will take about 45 min. While preparing the standard, gather the necessary materials for the experiment.

Sample preparation should be performed using the protocol for the specific human body sample source, ie, whole blood, bronchial wash, peritoneal fluid, etc.

Key of Abbreviations:

defRPMI          Serum Free deficient RPMI with HEPES (no biotin, phenol red, NaHCO3)
Staining media defRPMI + FCS + azide
Fixing media     defRPMI + 0.5% paraformaldehyde
hRPMI             RPMI with HEPES (NaHCO3 Free), (pink in color, NOT RPMI-1640)
Bimane media   hRPMI+FCS+azide+2.5mM probenicid
EtOH               Ethanol
MCB               Monochlorobimane
PF                    Paraformaldehyde
n                      Number of Samples
c                      Number of Compensation Samples

Preparation checklist:

Tube of MCB (10 mM, 100 µl in EtOH, brown eppendorf).
1 -2 tubes of frozen newborn calf serum (1 x n) ml required. Each tube contains 10 ml. Thawed and chilled in ice bath.
Tubes of antibodies:

If "n" is the number of samples to be stained on a particular day (n does not include standards), then to each tube add (53 x n)µl of premixed "GSH/PICU Stains". To each tube, add an additional (3 x n)µl of defRPMI. Keep tubes on ice. As an alternative, antibodies can be dispensed directly into the 96-well plates.
Compensation antibodies (FITC, PE, Cy5PE, Cy7PE, TR, APC or Cy5, Cy7APC, CasBlu, CasYel). Add directly to 96-well plate
Prelabeled 15 ml conical tubes: Prelabel 4 15 ml conical tubes with the appropriate coded labels for that day for each sample that will be used. Include 2 more tubes for the frozen standard.
2-4 96-well plates (flexible).
2 large ice buckets.
Bimane media (prepared fresh same day; pH to 7.4 final) Add Probenecid to 2.5 mM final (see below: Preparation of bimane staining media)

Room temperature: 400 ml as prepared (or 17 x n + 20 ml).
Ice cold: 400 ml as prepared (or 15 x n + 30 ml).

Fixing media. defRPMI + 0.5% PF. Make 3 x n + 2 ml. Use 4% PF stock (8x).
Staining media. defRPMI + FCS + azide. Make 1 x n mL.
FACS tubes in a rack:

One tube for each sample/staining combination.
One tube for each bimane stained FS (3 tubes).
One tube for unstained cells.
One tube for each compensation sample.

Carefully label all tubes with appropriate labels.

Preparation of bimane staining media:

Get out 2 or 4 bottles of hRPMI (stored in cold room). Warm hRPMI to 45C prior to adding probenecid to facilitate desolution. Use of a water bath is convenient. Temperatures as low as 37C may be used. 2 bottles is sufficient for approximately 5 samples.
Combine into bottle or beaker for adjusting pH, etc. To 400 ml, add:
16 mL FCS/HS mix
1 mL 10% sodium azide
0.285 g Probenecid
When probenecid is nearly in solution, pH to exactly 7.4. (Be sure solution is at RT before measuring pH)
Sterile filter into 500 ml receptacles: use 2, and divide evenly.
Put half (one bottle) in an ice-water slurry to cool, and leave the other at room temp. Label both bottles.

Preparation of staining/fixing media:

6. If defRPMI is to be made, refer to the recipe below this protocol. In our lab it is usually premixed and is stored in 250ml glass bottles in the cabinet to the right of the sink in the cold room.

7. Staining media: For each 200mL bottle of defRPMI, add 7mL of NCS, and 1mL of azide.

8. Fixing media (defRPMI + 0.5% PF): Make 50 mL on the day of use.. Make 1:8 dilution using 4% paraformaldehyde. It is best not to include serum in the fixing medium.

Preparation of Frozen Standard:

9. Chill centrifuge to 4C (with tube holders). Have bimane media at 4C. Set a water bath to 45C. Note 37C bath may be used.

10. Remove a frozen standard vial from liquid nitrogen and quickly transport to the lab. We are now testing the Frozen Standard 4 (prepared 3/27/98) stored in liquid nitrogen in Tank 1, 7B.
Note: Previous frozen standard 2 stored in 1, 3A. Three aliquots of frozen standard 1 remain in Tank 2, 7H and Tank 1, 3A, position 90.

11. Thaw sample immediately by gentle agitation at 45C until there is a small piece of ice remaining. Note 37C bath may be used.

12. Transfer to ice bath.

13. Slowly add 1 ml ice-cold bimane media drop by drop with micropipette. Pipette up and down with 1 ml pipetteman 3 times. Transfer to 15 ml tube. Slowly add 10-12 ml of ice-cold bimane media. Invert 3 times to mix.

14. Centrifuge at 1,200 rpm for 5 min.

15. Aspirate supernatant.

16. Resuspend pellet in 1 mL of bimane media and then add an additional 10-12 mL bimane media. Invert tube to mix.

17. Centrifuge at 1,200 rpm for 5 min.

18. Remove supernatant by aspiration; resuspend in 1.5 ml of cold bimane media. Remove 50 µl to Eppendorf tube and add 50 µl of Ethidium Bromide/Acridine Orange. Count live and dead cells. Record in log.

19. Return water bath to 37C.

TruCount Tubes preparation

1. Collect 2 x n TruCount tubes and note Lot number. For each sample, one tube will be used for the CD3/4/45 stain, and one will be used for the CD3/8/45 stain.

2. Add 20uL of prepared stains (found in box on Steve’s shelf in fridge 6) to the wall of each tube. Be careful NOT to resuspend the bead inside.

3. Make FACS Lysis solution. Make a 1:10 dilution in dH20. 450uL is needed for each tube. Make extra.

Sample preparation: See sample preparation for body fluid to be used.

Removing cells for TruCount and plasma Trx analysis

1. After removing 7mL for isolation of PBMC, take 40uL of whole blood and place in each TruCount tube (already containing stain). DO NOT resuspend beads in pipet. After adding blood, vortex lightly and let stand for 15 minutes.

2. After 15 minutes, add 450uL lysis buffer and put aside for FACS analysis (analysis can be done as many as 4 days after experiment)

3. Evenly distribute the remainder of blood into 1.5mL eppendorf tubes. Spin for 10 minutes at 14,000rpm in a microcentrifuge. Remove the plasma and repeat spin again at same conditions. Remove plasma and store in a screwcap tube in the -80C freezer

Isolation of PBMC from whole blood. 3 - 7 ml Green Top (Heparanized) blood is used.

1. 7 ml of whole blood is diluted with 4 ml of bimane media in a 15 ml conical centrifuge tube to achieve a total volume of 11 ml.. For pediatric and neonatal samples of smaller volumes, use 1:1 mix of blood and bimane media.

2. The diluted whole blood (11 ml) is overlaid onto 4 ml of room temperature Ficoll in a 15 ml conical centrifuge tube.

3. Samples are spun for 20 min at 2,000 rpm at room temperature. Be sure break on centrifuge is in the "Off" position.

4. With a sterile transfer pipette, remove the buffy layer and place into a sterile 15 ml conical tube. Dilute with 10 ml of bimane media. Dilute the frozen standard to about 10 ml with bimane media, and include this in the following spin.

5. Samples are spun for 10 min at 2,000 rpm, at room temperature.

6. The supernatant is aspirated, and the cells are resuspended in just over 1.1 ml of bimane media in the tube. Exactly 1.0 ml is transferred with a 1 ml pipetteman into a clean 15 ml conical centrifuge tube, and placed in the room temperature water bath for 5 min.

7. The frozen standard should be divided into three aliquots. To accomplish this, the supernatant is aspirated and the cells resuspended in about 3.5 ml of bimane media. Exactly 1.0 ml of the standard is transferred to three clean 15 ml tube (labeled FS-A, FS-B, FS-C) to be included in the MCB staining (steps 9 - 13). The remainder of the unstained frozen standard is kept on ice for later transfer to a FACS tube (see step 17).

8. Add 900 µl of bimane media to a pre-dispensed 100 µl MCB tube.

9. (Include the frozen standards in steps 9 - 13.) Add 40 µl of the diluted MCB to a sample every 15 seconds (concentration of MCB is now at 40uM). Use a stopwatch. Vortex each sample briefly after adding the MCB, and put back in the water bath. Cover with aluminum foil to block light; keep samples under cover from now on.

10. During 20 min incubation, cool down the centrifuge. Also, dispense antibodies into wells of 96-well trays, including the compensation stains (FITC, PE, CyC) (see diagram).

11. After 20 min incubation, quench the reactions in exactly the same order as the MCB was added, every 15 seconds: Add 10 ml ice-cold bimane media and transfer to ice.

12. Underlay each sample with about 1 ml of newborn calf serum.

13. Spin for 10 min at 2,000 rpm at 4C.

14. Aspirate the supernatant well. Resuspend sample cells in ((50 x n)+50)µl of bimane media.

15. For the MCB stained frozen standards, aspirate the supernatant well. Resuspend in 100 µl of bimane media and keep on ice for later transfer to FACS tubes containing 100 µl of defRPMI/0.5% PF (see step 19).

16. Put 50 µl of each sample into a well on the 96-well plastic tray. Keep the plastic tray in the aluminum block on ice at all times.

17. Combine the leftover cells from some of the sample tubes into one tube (for compensation samples). As an alternative method, another cell source can be use such as volunteer buffy cells. Resuspend sample cells in ((50 x c)+50)µl.

18. Once all samples are in the wells, use an 8-channel pipetter to transfer the antibodies to each sample quickly. Start the stopwatch after the last sample has been added. The objective here is to minimize the time difference that the samples see each stain pattern.

19. After the main Ab staining has begun, add about 50 µl of leftover cells (step 17) into each of the 3 wells containing the compensation stains. During the Ab staining, put the frozen standard samples, either stained with MCB or not, into the appropriate tubes containing defRPMI/PF.

20. After 15 min have elapsed, add 150 µl of cold bimane media to each well using multichannel pipetter.

21. Spin at 2,000 rpm for 3-5 min (do not centrifuge aluminum blocks). Always transfer immediately from centrifuge to blocks.

22. Aspirate wells; add 200 µl of cold bimane media. Repeat spin.

23. Repeat step 20 once more, for a total of 2 plus "one-half" washes and spins.

24. After the final aspiration, add 200 µl of defRPMI/0.5% PF to each well. Transfer to FACS tubes; keep under aluminum foil until all samples analyzed.

Special notes on MCB staining:

The MCB staining is subject to considerable intra-assay variation if not treated carefully.

A few points to remember:

1. Accurately pipette 40µl of bimane.

2. Accurately time the 20 min incubation, as indicated above.

3. After the reaction, add the 10 ml of cold media and immediately transfer onto ice. There is no exact method to stop the reaction, except for the cold temperature and this dilution.

4. After spinning with the newborn calf serum, aspirate very completely because any bimane that remains can continue to react.

5. After the bimane staining, always keep the cells on ice and cold - including transport to the centrifuge, and during transfer to FACS tubes. Remember to cool down the centrifuge and carriers during the 20 min bimane reaction.

Notes on Compensation:

It is important that compensation samples be chosen and stained properly. In multi-stain experiments, it will often be necessary to select multiple compensation samples for the same color. Our compensation samples only consist of one reagent, measured precisely and mixed with a fixed number of unstained cells, for each color of a multi-color stain. For each color, it is important to choose a compensation reagent that is as bright or brighter than your staining reagent. It is also desirable to use a compensation reagent that distinguishes clear positive and negative subsets to aid in determining the correct gates. For non-tandem dyes, using your brightest reagent for a particular color is desirable. For tandem dyes (Cy5PE, Cy7PE, Cy5.5PE, Cy7APC, Cy5.5APC), it is imperative that the compensation reagent be of the same tandem lot number, as different lots will vary in fluorescence (it must still be as bright, or brighter than your staining reagent). We typically choose compensation reagents that stain lymphocyte subsets (similar background of auto-fluorescence). If you need to choose a reagent that stains larger cells (such as monocytes), it may be useful to use the unstained monocytes from the unstained sample as your negative compensation control for this reagent. This controls for the higher auto-fluorescence on these larger cells. Collecting completely unstained cells is important in all experiments.

If you are doing a single stain experiment, the staining reagents for each color stained individually may suffice as your compensation reagents.

If CasBlu is the only color being excited by the UV laser, it is not essential that a CasBlu compensation sample be collected. However, you may wish to do so for quality control purposes.

If you are using MCB in your stain, you may not use any other reagents on Cascade Blue, as they are both excited by the UV laser at similar wavelengths.

Recipes:

MCB (monochlorobimane)

MCB (MW=226.66) is sold in 25mg bottles in powder form. We store MCB as frozen aliquots and use one aliquot each time we stain.

To prepare these frozen aliquots: Dissolve MCB powder in 11mL of 100% EtOH to make a 10mM solution. Aliquot 100uL accurately into brown Eppendorf tubes and keep in freezer.

NCS (newborn calf serum)

(1x n) mL required, thawed and chilled in ice bath

defRPMI

defRPMI consists of deficient RPMI (without biotin, riboflavin, phenol red, glutamine, or NaHCO3) with added HEPES at pH 7.0. See the Herzenberg Lab Media Preparation page for details.

hRPMI

To make hRPMI, please see our protocol Bimane Media (hRPMI) Formulation Protocol

Paraformaldeyhde (4%)

Paraformaldehyde is very toxic and aerates easily. Avoid breathing in the powder. Use a fume hood if necessary.

1. Mix required amount of paraformaldehyde (4g/100ml) to 2/3 final volume in ddH2O.

2. Heat to 60C while stirring in a fume hood (monitor temperature with thermometer).

3. Add 1 drop 2N NaOH to clear the solution.

4. Remove heat and add 1/3 vol 3x PBS.

5. Let cool and adjust to pH 7.2 with HCL.

6. Filter.