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One Step Fixation and Permeabilization
Materials: Maintain Reagents and buffers at 4°C.
- Fluorochrome-conjugated primary antibody
- Fluorochrome-conjugated isotype control antibody
- Fixation/Permeabilization Buffer, ADI-950-010
- Permeabilization Buffer, ADI-950-012
- PBS (Phosphate Buffered Saline), OmniPur 6505
- FBS (Fetal Bovine Serum), Gibco 10082
- 10 x 75mm, Tubes VWR 60818-281
Procedure:
- Harvest N x 106 cells and wash twice in 20 ml of PBS followed by centrifugation at 400 x g for 7 minutes (N=number of samples to be analyzed +1 for each isotype control).
- Re-suspend cells in N x 1 ml of Fixation/Permeabilization buffer.
- Mix well by inverting and incubate for 30 minutes at 4°C.
- Mix well by inverting and centrifuge at 400 x g for 7 minutes.
- Decant supernatant and re-suspend cells with N x 1 ml of Permeabilization Buffer.
- Centrifuge at 400 x g for 7 minutes.
- Decant supernatant and re-suspend pellet in N x 50 µl of Permeabilization Buffer.
- Gently mix by pipette and aliquot the cell suspension in 50 µl aliquots.
- Add the appropriate amount of antibody, to the cell aliquots, to achieve the target working concentration.
- Gently mix by pipette and incubate for 30 minutes in the dark at 4°C.
- Add 1 ml of Permeabilization Buffer.
- Gently mix by pipette and incubate for 5 minutes at 4°C.
- Centrifuge at 400 x g for 7 minutes.
- Add 1ml of Permeabilization Buffer.
- Gently mix by pipette and incubate for 5 minutes at 4°C.
- Centrifuge at 400 x g for 7 minutes.
- Decant supernatant and wash in 1 ml of PBS.
- Gently mix by pipette and centrifuge at 400 x g for 7 minutes.
- Decant supernatant and wash in 1 ml of PBS.
- Gently mix by pipette and centrifuge at 400 x g for 7 minutes.
-
Decant supernatant and gently re-suspend cell pellet by pipette with 500 µl of PBS containing 1% FBS for evaluation by flow cytometer.