Protocol

Reagents and Instruments

• Cell lines or primary cell cultures
• Phosphate buffered saline (0.01 M PBS pH 7.4)
• 70% ethanol
• Propidium iodide (PI)- Stock 400 μg/ml
• RNase- Stock 1mg/ml
• Triton X-100
• Polystyrene round bottom 12 x 75 mm Falcon tubes (Flow Tubes)
• 1 ml syringes with 26 G (Guage) needle
• Flow cytometer- FACSCalibur, Attune Nxt
• Softwares – CellQuest Pro, ModFit

Sample fixation

1. Dispense 1 x 10⁶ cells per ml of PBS in 5ml polystyrene tubes.
2. Wash the cells with 1 ml of PBS by centrifuging at 1000 rpm for 10 mins.
3. Fix the cells by adding 70% chilled ethanol drop wise to the cell pellet while vortexing. This should ensure fixation of all cells and minimize clumping.
4. Keep cells on ice for 1 hr or overnight at 4℃. (Fixed cells can be stored at 4℃ for 15-20 days)

Sample Staining

Procedure – I

1. Take 1 x 10⁶ fixed cells in 5ml polystyrene tubes and centrifuge at 1000 rpm for 10 mins.
2. Discard supernatant, wash the cells with 1 ml of PBS by centrifuging at 1000 rpm for 10 mins.
3. Add 500 μl of PBS to the cell pellet, vortex gently, add 100 μl of Propidium Iodide and 100 μl of RNase. Cover the tubes with aluminum foil to prevent its exposure to light. Incubate at 37ºC for 30 mins.
4. Pass the sample through 26 G needle to prepare single cell suspension.
5. Acquire the sample on FACSCalibur / Attune NxT.

Procedure- II

1. Take 1 x 10⁶ fixed cells in 5ml polystyrene tubes and centrifuge at 1000 rpm for 10 mins.
2. Discard the supernatant and wash the cells with 1 ml of PBS by centrifuging at 1000 rpm for 10 mins
3. Re-suspend the pellet in 500 μl of PBS containing 0.1% Triton X-100 and 100 μg/ml of RNaseA for permeabilization and RNase inactivation respectively. Incubate at 37ºC for 15 mins.
4. After incubation, add 25 μg/ml of propidium iodide to the cell suspension, mix properly and further incubate at 37°C for 15 minutes.
5. Pass the cell suspension through 26 G needle to avoid clumping of sample.
6. Acquire the sample on FACSCalibur / Attune NxT.

Preparation of reagents

1. Phosphate buffer saline (0.01 M PBS pH 7.4)

Dissolve in 800 ml distilled water (dH₂O)
Adjust the pH to 7.4 with HCl
Finally, make up the volume to 1 L with dH₂O.
Store at 4°C.

2. 70 % ethanol

Store at 4°C.

3. Propidium iodide (PI): Stock – 400 μg /ml

Dissolve and Aliquots in 1.5 ml Eppendorf tubes
Store at -20°C.
(Precaution: Cover the 1.5 ml Eppendorf tubes with Aluminum foil as PI is light sensitive.

4. RNase: Stock – 1 mg/ml

Dissolve and aliquot in Eppendorf tubes.
Store at -20°C.

Point to remember:

1. The quality of sample should be maintained for better result.
2. Try to make single cell suspension before fixation of the sample to avoid clumping (to prevent the doublets).
3. Handle the tubes with care and wear gloves to avoid contact with skin as PI is mutagenic.

There are three general staining protocols based on type of antigen to be evaluate, nature of Fluorochrome and sample type.

1. Direct Staining:

   This technique involves incubation of cells with fluorochrome conjugated antibody, which recognize and bind to various cell surface antigens. The antibodies are mostly monoclonal antibodies (MAb) and they may be added either single or in multiple combinations depending upon the analysis.

2. Indirect staining:

   In indirect staining technique, the cells are incubated with an unconjugated primary antibody. The primary antibody recognizes and binds to cell surface antigens then the cells are subsequently incubated with a fluorochrome conjugated secondary antibody.

3. Intracellular staining:

   This technique involves detection of intracellular antigens; it done by direct staining method using conjugated antibodies. Cells are initially subjected to a fixation step with paraformaldehyde in order to minimize leakage of proteins out of the cell, following permeabilization by mild detergents such as saponin or Tween 20. The permeabilization allows access of conjugated antibodies to proteins within the cell. The conjugated antibody penetrates and binds to its target within the cell. Intracellular staining technique is very useful in monitoring immune responses and tracking signalling molecules.

Reagents and Instruments

• Cell line or primary cells
• Phosphate buffered saline (PBS, 0.01M pH 7.4)
• FACS Buffer I (for surface staining)
• FACS Buffer II (for intracellular staining)
• 1% Paraformaldehyde
• Refrigerated centrifuge
• Polystyrene round bottom 12 x 75 mm Falcon tubes
• Micropipettes of capacity- 1 ml, 200 μl, 10 μl
• Flow cytometer – FACSCalibur, Attune NxT or FACSAria
• Software - CellQuest Pro (FACSCalibur), Attune NxT (Attune NxT), FACSDiva (FACSAria)

Antibodies

Direct staining:
1. Fluorochrome labelled antibody
2. Isotype matched fluorochrome labelled antibody
Indirect staining:
1. Unlabelled primary antibody
2. Fluorochrome labelled secondary antibody

Procedure

1. Direct Staining

   1. Dispense 1 x 10⁶ Cells in 5ml polystyrene tubes.
   2. Add 1 ml of FACS buffer I and centrifuge the tubes at 1000 rpm for 10 mins at 4°C. Discard the supernatant. (washing)
   3. Resuspend the cells in 100 μl of FACS buffer I.
   4. Add appropriate amount of antibody (the amount of antibody can be decided as per manufacturer’s recommendation and titration)
   5. As negative control, incubate the cells with isotype matched antibody conjugated with same fluorochrome used for the test.
   6. Incubate the cells on ice at 4°C for 45 mins. in dark.
   7. Wash the cells 2 times and finally resuspend in 500 μl of FACS buffer I.
   8. Acquire the sample on flow cytometer as soon as possible.
   9. If the acquisition is not possible on the same day, fix the cells in 500 μl of 1% paraformaldehyde to preserve the cells for several days (this will stabilize the light scatter, prevent quenching and inactivate most biohazardous agents).

2. Indirect Staining

   1. Dispense 1 x 10⁶ Cells in 5ml polystyrene tubes.
   2. Add 1 ml of FACS buffer I and centrifuge the tubes at 1000 rpm for 10 min at 4°C. Discard the supernatant (washing).
   3. Resuspend the cells in 100 μl of FACS buffer I.
   4. Add appropriate amount of unconjugated purified monoclonal antibody specific for antigen to be detected. (the amount of antibody can be change based on manufacturer’s recommendation and titration)
   5. Incubate on ice at 4 ⁰ C for 45 mins.
   6. Wash the cells 2 times with FACS buffer I as described before to remove excess antibody.
   7. Dilute the fluorochrome- labelled secondary antibody as per manufacturer’s instruction in FACS buffer I.
   8. Incubate the cells with diluted secondary antibody (specific for primary antibodies) conjugated with fluorochrome on ice at 4⁰ C for 45 min.
   9. As negative controls, incubate the cells with isotype matched controls conjugated with same fluorochrome used for the test.
   10. Wash the cells 2 times and finally resuspend in 500 μl of FACS buffer I.
   11. Acquire the sample on flow cytometer as soon as possible.
   12. If the acquisition is not possible on the same day, fix the cells in 500 μl of 1% paraformaldehyde to preserve the cells for several days (this will stabilize the light scatter, prevent quenching and inactivate most biohazardous agents).

3. Intracellular staining

   1. Dispense cells in 5ml tubes.
   2. Add 1 ml of FACS buffer I and centrifuge the tubes at 1000 rpm for 10 mins at 4⁰C. Discard the supernatant (washing).
   3. Add 500 μl of 1 % paraformaldehyde to fix the cells.
   4. Incubate on ice at 4⁰C for 15 mins. with intermediate mixing of the tubes.
   5. Wash the cells with FACS buffer I by centrifuging tubes at 1000 rpm for 10 mins. at 4⁰C twice.
   6. After second wash, Add 100 μl FACS buffer II to the cell pellet to permeabilize the cells.
   7. Gently vortex the tubes.
   8. Incubate at room temperature for 5 min.
   9. Add recommended amount of fluorochrome labelled antibody specific for antigen to be detected.
   10. As negative control, incubate the cells with isotype matched controls conjugated with same fluorochrome used for the test.
   11. Incubate on ice at 4°C for 45 mins. in dark.
   12. Wash the cells 2 times and resuspend in 500 μl of FACS buffer I.
   13. Acquire the sample on flow cytometer.

Preparation of reagents:

1. Phosphate buffer saline (0.01 M PBS pH 7.4)

Dissolve in 800 ml distilled water (dH₂O)
Adjust the pH to 7.4 with HCl
Finally, make up the volume to 1 L with dH₂O.
Store at 4°C.

2. FACS buffer I

Store at 4°C.

3. FACS buffer II

Store at 4°C.

4. 1 % paraformaldehyde

Prepare solution in a glass bottle. Heat in microwave oven until the powder dissolves completely. Allow it to cool. Cover the bottle with Aluminium foil to prevent exposure to light. Store at 4°C.
(Precaution: Do not inhale the fumes)

Point to remember:

1. Before starting the experiment, the titration of antibodies should be done according to manufacturer’s instruction for optimum concentration.
2. The recommended controls should be run with every experiment for better analysis e.g. Iso control
3. In case of multicolour analysis use compatible compensation controls.

Reagents and Instruments:

• Phosphate buffered saline (0.01 M PBS pH 7.4)
• Annexin-V FITC
• Propidium iodide (PI) – stock- 400 μg/ml
• Annexin Binding buffer- 10X
• Refrigerated centrifuge
• Polystyrene round bottom 12 x 75 mm Falcon tubes
• Micropipettes of capacity- 1 ml, 200 μl, 10 μl
• Flow cytometer- FACSCalibur, Attune Nxt
• Softwares – CellQuest Pro, Attune NxT, Flow Jo

Procedure:

1. Wash the cells twice with 1X PBS and then resuspend in 1X Annexin Binding Buffer at a concentration of 1 X 10⁶ cells/ml.
2. Transfer 100 μl of the cell suspension (1 X 10⁵ cells) in another polystyrene tube.
3. Add 2-5 μl of Annexin V FITC.
4. Add 40-50 μg of Propidium Iodide.
5. Gently vortex the cells and incubate for 15 min at room temperature in the dark.
6. Add 300 μl of 1X Annexin Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.

The following controls are used to set up compensation

1. Unstained cells.
2. Cells stained with only Annexin V FITC
3. Cells stained with only Propidium Iodide

Preparation of Reagents:

1. Phosphate buffer saline (0.01 M PBS pH 7.4)

Dissolve the following in 80ml MilliQ Water:

2. Annexin binding buffer 10X

Adjust the pH to 7.4. Make up the volume to 100 ml with MilliQ water. Store at 2-8°C

3. Propidium iodide (PI): Stock – 400 μμg /ml

Dissolve and aliquot in 1.5 ml capacity tubes.
Store at -20°C.
(Precaution: Cover the tube with aluminium foil as PI is photo-sensitive.)

Point to remember:

1. Before starting the experiment, the titration of Annexin V should be done to decide optimal concentration.
2. The pH of Annexin binding buffer should be maintained for better results.
3. Handle with tubes with care and wear gloves to avoid contact with skin as PI is mutagenic.