Intracellular Flow Cytometry Kit (Triton™ X-100) #51995

Intracellular Flow Cytometry Kit (Triton™ X-100)

NOTE: Prior to using this kit, refer to the product webpage for antibodies of interest to determine whether they are validated for Flow Cytometry (F) assays using a Triton™ X-100 permeabilization protocol.

NOTE: When used in conjunction with fluorescent cellular dyes (including viability dyes, DNA dyes, etc.), please refer to the dye product page for recommended protocol. Visit www.cellsignal.com for a listing of flow cytometry validated cellular dyes.

A. Solutions and Reagents

Supplied Reagents:

• 10X Wash Buffer, Phosphate Buffered Saline (PBS) (#12528): Dilute desired amount to a 1X working solution with reverse osmosis deionized (RODI) or equivalent grade water, mix. Store at 4°C and use within one month once diluted.
• 4% Formaldehyde, methanol-free (#47746): Store at room temperature. Use within one month once opened.
• Cell Permeabilization Buffer (Triton™ X-100) (#39487): Store at 4°C.
• Flow Cytometry Antibody Dilution Buffer (#13616): Store at 4°C.

Additional Reagents (Not Supplied):

• Red blood cell lysis buffer
• Unconjugated or fluorochrome-conjugated primary antibodies
• Fluorochrome-conjugated secondary antibodies (if applicable)
• Cellular dyes (e.g., live-dead discrimination, DNA dyes, etc.), optional

NOTE: Not all extracellular antibodies will bind correctly after fixation and/or detergent permeabilization, so a small-scale experiment should be performed if you are unsure. Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation. The antibodies will remain bound to the target of interest during the fixation process. A wash step prior to fixation may be performed but is not necessary.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

1. Pellet cells by centrifugation and remove supernatant.
2. Resuspend cells in approximately 100 µl 4% Formaldehyde (#47746) per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
3. Fix for 15 min at room temperature (20-25°C).
4. Wash by centrifugation with excess 1X Wash Buffer. Discard supernatant containing formaldehyde in appropriate waste container. Repeat.
5. Proceed to Permeabilization step, or store overnight in 1X PBS at 4°C.

C. Permeabilization

1. If necessary, pellet cells by centrifugation and aspirate supernatant.
2. Permeabilize cells by adding 100 µL Cell Permeabilization Buffer (Triton™ X-100) (#39487) per 1 million cells.
3. Permeabilize for a minimum of 10 minutes.
4. Proceed to Immunostaining step, or store cells overnight in 1X PBS at 4°C.

D. Immunostaining

1. Aliquot desired number of cells into tubes or wells. (Generally, 5 x 10⁵ to 1 x 10⁶ cells per assay.)
2. Pellet cells by centrifugation and remove supernatant.
3. Resuspend cells in 100 µl of diluted antibody or antibody conjugates, prepared in Flow Cytometry Antibody Dilution Buffer (#13616) at the recommended dilutions. See individual antibody datasheets or product webpage for recommended dilution, or determine via titration.
4. Incubate for 1 hr at room temperature (20-25°C).
5. Wash by centrifugation in excess 1X Wash Buffer. Discard supernatant. Repeat.
6. If using a fluorochrome-conjugated primary antibody, resuspend cells in 200-500 µl 1X Wash Buffer and analyze on flow cytometer; for unconjugated primary antibodies, proceed to next step.
7. Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in Flow Cytometry Antibody Dilution Buffer (#13616) at the recommended dilution.
8. Incubate for 30 min at room temperature (20-25°C).
9. Wash by centrifugation in excess 1X Wash Buffer. Discard supernatant. Repeat.
10. Resuspend cells in 200-500 µl 1X Wash Buffer and analyze on flow cytometer.