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 |