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Bethyl Flow Cytometry Protocol

Flow Cytometry Protocol for Indirect Intracellular Staining of Cultured Cells Grown in Suspension.

Reagents Required:
16% parafolmaldehyde (PFA) (Electron Microsopy Sciences RT 15710)
90% Methanol (ice cold)
PBS (Phosphate Buffered Saline pH 7.2)
11.6 g sodium chloride
10 ml 1 M phosphate buffer
0.26 Ml 4 M sodium hydroxide
DI H2O to 1 liter
ICSB (IntraCellular Staining Buffer)
495 ml PBS
5 ml FBS (feteal bovine serum)
0.36 ml 9% soldiuim azide
12 x 75 FACS tubes (BD Falcon #352054)

Cell Fixation and Permeabilization:
  1. For cells grown in suspension, add fresh 16% PFA to achieve a final concentration of 1.54% directly to the cells in media (1-2 x 10^6 cell/ml).
  2. Fix cells at room temperature (RT) for 10 minutes.
  3. Incubate and fix on ice for an additional 30 minutes.
  4. Spin fixed cells at 250 x g for 5 minutes at 20 °C.
  5. Pour off media + PFA into dedicated PFA waste containier.
  6. Permeabilize cells by resuspending in ice cold 90% methanol to a final cell concentration of 1 X 10^6 cells/ml.
  7. Cells may be stored at -70 °C to -20 °C for up to a month.

Intracellular Staining:

  1. Aliquot 1 ml (1 x 10^6 cells) of cells in methanol for each tube/sample.
  2. Spin at 250 x g for 5 minutes at 20 °C, break set to slow (perform all subsequent spins at these conditions)
  3. Pour off methanol.
  4. Resuspend cells in 1 ml ICSB for wash.
  5. Pour off wash and blot tube on paper towels.
  6. Resuspend cells in 50 mcl of primary antibody at desired dilution.
  7. Incubate at RT for 1 hour.
  8. Add 1 ml of ICSB.
  9. Spin.
  10. Pour off and blot tube.
  11. Resuspend in 100 mcl of conjugated secondary antibody at desired dilution.
  12. Incubate 30 minutes at RT in dark.
  13. Add 1 ml ICSB for 2nd wash.
  14. Spin, pour off, and blot tube.
  15. Resuspend in 100 mcl ICSB.
  16. Analyze on a flow cytometer.

Flow Cytometry: Protocols, Kits and Reagents

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