flow cytometry protocol

Direct immunostaining of surface antigens

Key Reagents – PBS, staining buffer, FACS buffer, PFA fixing buffer

• Prepare a single-cell suspension using the appropriate protocol and adjust the cell concentration to 107 cells/ml in staining buffer
• Dispense 100µl of cell suspension into as many staining tubes as needed [unstained control, compensation controls, optional isotype, and FMO controls, and test sample].
• Add the optimized dilution of antibodies to the respective tubes and incubate at 4°C (on ice) for 30 minutes in the dark.
• Wash the cells once with ice-cold PBS at 300-400 x g and resuspend in 100-200µl FACS buffer/PFA fixing buffer.
• Store at 4°C in darkness and acquire preferably within 24 hours.

Indirect immunostaining of surface antigens

Key Reagents – PBS, staining buffer, FACS buffer, PFAfixing buffer

• Prepare a single cell suspension using the appropriate protocol and adjust the cell concentration to 107 cells/ml in staining buffer.
• Dispense 100µl of cell suspension into as many staining tubes as needed [unstained control, compensation controls, optional isotype and FMO controls, and test sample].
• Add the optimized dilution of primary antibodies to the respective tubes and incubate at 4°C (on ice) for 30 minutes.
• Wash the cells once with ice cold PBS at 300-400 x g and re-suspend in 100µl staining buffer.
• Add the specific secondary antibodies at the proper dilution and incubate the cells at 4°C (on ice) for 30 minutes in the dark.
• Wash the cells once with cold PBS at 300-400 x g and re-suspend in 100-200µl FACS buffer/PFA fixing buffer.
• Store at 4°C in darkness and acquire preferably within 24 hours.

General immuno-staining procedure for intracellular antigens

Key Reagents – PBS, staining buffer, FACS buffer, 0.5-4% PFA in PBS [exact concentration of PFA has to be standardized for every antibody panel], 100% methanol

A. Permeabilization with methanol

• Perform surface staining as per protocols 1 or 2 along with the suitable controls.
• Aliquot the stained cells in 0.5-4% PFA at 107 cells/ml. Prepare unstained aliquots for the intracellular staining controls.
• Fix the cells on ice for 10-30 minutes away from light.
• Wash out the fixative at 300-400 x g and slowly add ice-cold 100% methanol with gentle vortexing.
• Incubate the cells on ice for 30 minutes away from light.
• Wash out the methanol at 400-500 x g and re-suspend the cells in 100µl staining buffer.
• Dispense 100µl of cell suspension into as many staining tubes as needed [unstained control, compensation controls, optional isotype and FMO controls, and test sample].
• Add the optimized dilution of primary antibodies to the respective tubes and incubate at 4°C (on ice) for 30 minutes.
• Wash the cells once with ice cold PBS at 400-500 x g and re-suspend in 100µl staining buffer.
• Add the specific secondary antibodies at the proper dilution and incubate the cells at 4°C (on ice) for 30 minutes in the dark.
• Wash the cells once with cold PBS at 400-500 x g and re-suspend in 100-200µl FACS buffer/PFA fixing buffer.
• Store at 4°C in darkness and acquire preferably within 24 hours.

B. Permeabilization with saponin

Key Reagents – PBS, staining buffer, FACS buffer, 0.5-4% PFA in PBS [exact concentration of PFA has to be standardized for every antibody panel], 0.1% saponin

• Perform surface staining as per protocols 1 or 2 along with the suitable controls.
• Aliquot the stained cells in 0.5-4% PFA at 107 cells/ml. Prepare unstained aliquots for the intracellular staining controls.
• Fix the cells on ice for 10-30 minutes away from light.
• Wash out the fixative at 300-400 x g and add 0.1% saponin.
• Incubate the cells at room temperature for 15 minutes.
• Wash out saponin at 300-400 x g and re-suspend the cells in 100µl staining buffer.
• Dispense 100µl of cell suspension into as many staining tubes as needed [unstained control, compensation controls, optional isotype and FMO controls, and test sample].
• Add the optimized dilution of primary antibodies to the respective tubes and incubate at 4°C (on ice) for 30 minutes.
• Wash the cells once with ice cold PBS at 400-500 x g and re-suspend in 100µl staining buffer.
• Add the specific secondary antibodies at the proper dilution and incubate the cells at 4°C (on ice) for 30 minutes in the dark.
• Wash the cells once with cold PBS at 400-500 x g and re-suspend in 100-200µl FACS buffer/PFA fixing buffer.
• Store at 4°C in darkness and acquire preferably within 24 hours.

Intracellular cytokine/phospho-immunostaining

Key Reagents – PBS, staining buffer, FACS buffer, 0.5-4% PFA in PBS [exact concentration of PFA has to be standardized for every antibody panel], 100% methanol

The intracellular staining procedure allows direct measurement of antigens (cytokines or transcription factors) present inside the cytoplasm or in the nucleus of a cell in addition to the surface antigen determination simultaneously. In this procedure, the fixation and permeabilization of cells are required after staining with fluorescently conjugated surface antigens. This modified staining procedure allows direct measurement of functional activity of any cell of interest present in the blood or other tissues without further separation. To achieve better results, additional in vitro cell stimulation with some common mitogen (e.g., PMA, Ca++ or peptide epitopes and protein transport inhibitor, Brefeldin A, etc.) may be required, which allows increased production of cytokines inside cells. Refer to the table below as a guideline for common cell stimulation procedures.

Note: If the cells are stained in a 96 well U- or V-bottom plate, washing procedure should be set up first for maximum removal of unbound primary or secondary antibodies.

• Harvest cells using the suitable protocol and aliquot them in tubes at the pre-determined concentration (depending on cell type and stimulant).
• Add the specific stimulant and incubate the cells at 37°C for the requisite time. The table below is a handy reference of different stimulants and incubation time vis-à-vis the target proteins.
• In the case of staining for secreted cytokines, add brefeldin A or monensin during the incubation period at the concentration recommended by the manufacturer.
• Set aside some unstimulated aliquots for the unstained control and stained baseline controls.
• Stop the stimulation by fixing the cells with the final concentration of 0.5% PFA.
• Vortex gently and keep cells on ice for 15 minutes.
• Wash off the fixative and proceed with surface staining as per protocol1 or 2.
• Re-suspend cells in the preferred permeabilizing reagent and proceed with the permeabilization and intracellular staining accordingly as per protocol 3a or 3b (100% methanol or 0.1% saponin).

In Vitro Cell Stimulation Reference Table

Target cytokine/phosphoprotein	Target cells	Stimulant	Duration	Surface marker
IL-2	PBMCs	PMA (50ng/ml)	4-6 hours	CD3
IL-3	T-cells	PMA(50ng/ml) + ionomycin (1µg(ml)	4-6 hours	CD4
IL-4	PBMCs	PMA(50ng/ml) + ionomycin (1µg(ml)	4-6 hours	CD4
IL-6	PBMCs	LPS (100ng/ml)	4-6 hours	CD14
IL-10	PBMCs	LPS (100ng/ml)	18-24 hours	CD14
GM-CSF /IFNγ/TNFα/TNFβ	PBMCs	PMA(50ng/ml) + ionomycin (1µg(ml)	4-6 hours	CD3
pStat5	PBMCs	GM-CSF (20ng/ml) + IL3 (20ng/ml)	15 min.	CD123, CD116
pStat3	PBMCs	G-CSF (20ng/ml) + IL6 (20ng/ml)	15 min.	CD126, CD114
pERK	PBMCs	IL3 (20ng/ml) + IL6 (20ng/ml) + FLT3L (20ng/ml)	15 min.	CD123, CD126, CD135

Dye efflux staining

Dye exclusion staining is performed to separate live and dead cells, as well as to isolate the rare stem cell ‘side populations’. If viability staining is included in your regular immunostaining, it should be performed before any other staining.

A. Propidium iodide (PI) staining (viability)

Key reagents – PBS, staining buffer, PI solution (10µg/ml in PBS)

• Harvest the cells and wash once with PBS.
• Re-suspend cells in staining buffer at 107 cells/ml.
• Add 5µl of PI stain per 100µl of cell suspension, mix gently, and let it stay in the dark for 1 minute.
• Wash out the dye and re-suspend cells in a suitable volume of staining buffer.

B. 7-Amino actinomycin D (7-AAD) staining (viability)

Key reagents – PBS, staining buffer, 7-AAD solution (100µg/ml in PBS)

• Harvest the cells and wash once with PBS.
• Re-suspend cells in staining buffer at 10⁷ cells/ml.
• Add 2µl of 7-AAD stain per 100µl cell suspension, mix gently and incubate the cells on ice for 30 minutes.
• Wash out the dye and re-suspend cells in a suitable volume of staining buffer.

C. Rhodamine 123 or Hoechst 33342 staining (side population)

Key reagents – PBS, 5% FBS in PBS, staining buffer, Hoechst 33342 solution (1mM in PBS) or Rho123 solution (10µg/ml in PBS)

• Harvest the cells and wash them once in PBS.
• Re-suspend cells in 5% FBS at 10⁷ cells/ml.
• Add 1µl of the dye per 100µl of cell suspension and incubate in a water bath at 37°C for 30 minutes.
• Wash out the dye, re-suspend the cells in pre-warmed, dye-free 5% FBS, and repeat above the incubation step.
• Wash the cells again and re-suspend in a suitable volume of staining buffer

DNA content or Cell cycle analysis

Key reagents – PBS, staining buffer, PI solution (50µg/ml in PBS), RNAse A (10µg/ml), 70% ethanol

• Harvest cells and wash once in PBS.
• Re-suspend cells in staining buffer at 107 cells/ml.
• Aliquot 500µl of cells into separate tubes (pre-chilled) and add ice cold 70% ethanol dropwise with gentle vortexing.
• Keep cells on ice for 1 hour.
• Wash the cells twice in PBS at 400-500 x g for 10 minutes.
• Add 1ml of PI solution to the cell pellet and mix well. Add 50µl of RNase to a final concentration of 0.5µg/ml.
• Incubate the cells at 4°C overnight.
• Wash once in PBS and re-suspend in a suitable volume of staining buffer.