Flow Cytometry, Methanol Fixation

A. Solutions and Reagents

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

1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
2. 100% Methanol (#13604): Chill before use.
3. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

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

B. Fixation

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

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by methanol. The antibodies will remain bound to the target of interest during the fixation process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to fixation. Conduct a small-scale experiment if you are unsure.

1. Pellet cells by centrifugation and remove supernatant.
2. Fix cells by adding ice-cold 100% methanol slowly, while gently vortexing. Add sufficient volume to achieve 90% methanol (accounting for residual liquid remaining after centrifugation), and additional methanol if needed to reach at least 100 µl per 1 million cells.
3. Fix for 15 min at -20 °C. Methanol will also permeabilize the cells at this time, so no additional permeabilization step is necessary.
4. Proceed to Immunostaining step.
   1. Alternatively, cells may be stored in methanol at -20°C.

C. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

1. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
2. Aliquot desired number of cells into tubes or wells. (Generally, 5x10⁵ to 1x10⁶ cells per assay.) Pellet cells by centrifugation and remove supernatant.
3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration. Mix well to dissociate cell pellet.
4. Incubate for 1 hr at room temperature.
5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat. If using directly conjugated antibodies, skip to step 9.
6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
7. Incubate for 30 min at room temperature. Protect from light.
8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
9. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.