mTOR (7C10) Rabbit mAb (Alexa Fluor^® 647 Conjugate) #5048

Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

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.
4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

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

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

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

1. Aliquot desired number of cells into tubes or wells.
2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
4. Incubate for 1 hr at room temperature. Protect from light.
5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
2. Incubate for at least 5 min at room temperature.
3. Analyze cells in DNA staining solution on flow cytometer.