Flow Cytometry Protocol (Flow)

IMPORTANT: Please refer to the APPLICATIONS section on the front page of product datasheet or product webpage to determine if this product is validated and approved for use in Flow Cytometry (F). Please see the product-specific Flow protocol on the product webpage for appropriate fixation and permeabilization conditions, and recommended antibody dilution.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix.
2. 16% Formaldehyde (methanol free).
3. 100% methanol (if required).
4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
5. Secondary Antibodies: Anti-mouse (#4408, #8890, #4410, #8887), Anti-rabbit (#4412, #8889, #4414, #8885), Anti-rat (#4416, #4418)

B. Fixation

NOTE: If live cell staining is desired, proceed to Immunostaining (Section D). Please refer to the product webpage and product-specific protocol to determine whether it is compatible with live cell staining.

1. Collect cells by centrifugation and aspirate supernatant.
2. Resuspend cells in 0.5–1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
3. Fix for 15 min at room temperature.
4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

NOTE: Please refer to the product-specific protocol on the product webpage for correct permeabilization conditions. Not all products are compatible with methanol permeabilization.

1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
2. Incubate 30 min on ice.
3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

1. Aliquot desired number of cells into tubes or wells.
2. Wash cells by centrifugation in excess 1X PBS to remove methanol, if required. Discard supernatant in appropriate waste container. Repeat if necessary.
3. Resuspend cells in 100 µl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
4. Incubate for 1 hr at room temperature (fixed cells) or 15-30 minutes on ice (live cells).
5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat. If using a directly conjugated antibody, skip to step 9.
6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in incubation buffer at recommended dilution).
7. Incubate for 30 min at room temperature (fixed cells) or on ice (live cells).
8. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
9. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining or live/dead discrimination, proceed to optional DNA stain (Section E).

E. Optional DNA Dye (fixed cells) or Live/Dead Discrimination (live cells)

1. Resuspend cells in 0.5 ml of DNA dye or live/dead discriminator dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
2. Incubate for 5 min at room temperature (fixed cells) or on ice (live cells).
3. If required, wash by centrifugation in PBS. Analyze cells on flow cytometer.