Flow Cytometry (FC) Protocol

Collect cells and make a single-cell suspension. Filter clumps if needed. Count cells and transfer 0.5-1 x 10^6 cells per tube or well. Keep cells on ice or at 4 degrees C when staining sensitive surface markers.

Add 1-2 mL staining buffer per tube, or 150-200 uL per 96-well plate well. Staining buffer can be PBS or HBSS with 1-2 percent FBS or BSA and 1-2 mM EDTA. Centrifuge 300-500 x g for 5 minutes and discard supernatant.

Resuspend cells in 50-100 uL staining buffer containing Fc block or 5-10 percent normal serum when working with Fc receptor-positive cells. Incubate 10-15 minutes at 4 degrees C.

Add primary antibody at the datasheet-recommended flow cytometry amount or start with 0.25-1 ug per 10^6 cells for unconjugated antibody. For conjugated antibodies, use the recommended test size or titrate 0.125x, 0.25x, 0.5x, 1x, and 2x. Final staining volume is commonly 50-100 uL.

Incubate 20-30 minutes at 4 degrees C protected from light. Mix gently halfway through if cells settle.

Add staining buffer, centrifuge 300-500 x g for 5 minutes, and discard supernatant. Wash 2 times. Resuspend final pellet in 200-500 uL staining buffer for acquisition.

If using an unconjugated primary antibody, add fluorophore-conjugated secondary antibody after the first wash. Incubate 20-30 minutes at 4 degrees C protected from light, then wash 2 times.

Filter samples if clumps are present. Acquire enough events for the population of interest. Use viability dye, unstained cells, single-color controls, fluorescence minus one controls, and isotype controls when appropriate.

Prepare 0.5-1 x 10^6 cells per tube or well. If surface markers are also needed, stain surface antigens first using the surface staining protocol before fixation unless the surface antibody is fixation-resistant.

Add fixation buffer according to the kit or reagent instructions. A common starting condition is 1-4 percent paraformaldehyde for 10-20 minutes at room temperature. Wash with staining buffer.

Resuspend cells in permeabilization buffer, such as saponin-based buffer for many cytoplasmic targets or methanol/permeabilization kit buffer for nuclear and phospho targets. Incubate 10-30 minutes as recommended.

Incubate cells in permeabilization buffer containing 1-5 percent serum or BSA for 10-20 minutes. Keep cells in permeabilization buffer during antibody incubation and washes when using saponin-based systems.

Dilute primary antibody in permeabilization buffer. Start with the datasheet-recommended flow dilution or titrate 0.25-2 ug per 10^6 cells. Incubate 30-60 minutes at room temperature or 4 degrees C, protected from light when fluorophores are present.

Wash 2 times with permeabilization buffer. If the primary antibody is unconjugated, add fluorophore-conjugated secondary antibody for 20-30 minutes, then wash 2 times.

Resuspend cells in 200-500 uL staining buffer. Acquire samples promptly or store fixed stained cells at 4 degrees C protected from light for a short period if compatible with the fluorophores and assay.

Set up untreated, stimulated, inhibited, and positive control samples. Time stimulation carefully because phosphorylation can change within minutes.

At each time point, fix cells quickly to preserve signaling state. Add pre-warmed fixation buffer to reach the recommended final concentration, commonly 1-4 percent paraformaldehyde, and incubate 10-15 minutes.

Wash cells, then permeabilize using cold 90 percent methanol added slowly while vortexing gently, or use a phospho-flow permeabilization kit. Incubate on ice or at -20 degrees C for 10-30 minutes depending on the protocol.

Wash cells 2 times with staining buffer to remove methanol. Block in staining buffer with 1-5 percent BSA or serum for 10-20 minutes.

Dilute primary antibody in staining buffer. Use the datasheet-recommended flow dilution or titrate across a range. Incubate 30-60 minutes at room temperature or 4 degrees C.

Wash 2 times. Add fluorescent secondary antibody if needed and incubate 20-30 minutes protected from light. Wash again and resuspend for acquisition.

Use unstimulated and stimulated controls to set gates. Compare median fluorescence intensity rather than percent positive when signal shifts are continuous.

Assign bright fluorophores to low-abundance markers and dimmer fluorophores to high-abundance markers. Avoid placing highly co-expressed markers in channels with severe spillover.

Titrate each antibody individually using the same cell type and staining conditions planned for the experiment. Choose the lowest antibody amount that gives clear separation with acceptable background.

Aliquot 0.5-1 x 10^6 cells per sample. Wash and block Fc receptors for 10-15 minutes at 4 degrees C.

Prepare a master mix containing all antibodies at final optimized concentrations. Include primary antibody if it is part of the panel. Add the cocktail to cells in 50-100 uL final staining volume and incubate 20-30 minutes at 4 degrees C protected from light.

Wash 2 times with staining buffer. Fix cells if required by biosafety policy or if acquisition will be delayed. Confirm that fluorophores tolerate fixation.

Prepare unstained cells, single-color compensation controls, viability-only control, and fluorescence minus one controls for critical gates. Include biological positive and negative controls when available.

Acquire enough events for rare populations. Use compensation controls to correct spillover and FMO controls to place gates for dim or continuous markers.