Measurement of lipid raft localization via flow cytometry
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| Materials | |
|---|---|
| PBS (pH 7.4)
Trypsin/EDTA Triton-X100 Serum starvation medium: 0.5% calf serum (CS) FACS buffer: 46.5 ml PBS + 2.5 ml FCS (h.i) + 1 ml 5% sodium azide |
Procedure
- Add cells in poly-L-lysin coated dishes
- Optionally: Serum starvation of cells over night, 37 °C
- Positive control: fluorescently labelled choleratoxin for staining of Gm1
- Negative controls: staining of transferrin receptor, untransfected cells without staining
- 1st antibody 1:1000 in 3 ml medium for 1h, 37 °C
- 2x wash with PBS
- 2nd antibody 1:1000 or choleratoxin 1:400 in 3 ml medium, 30 min, 37 °C
- Dissolve cells with trypsin/EDTA or cell scrachter
- 2x wash with PBS
- Divide cell solution in two aliquots
- Analyze first aliquot via flow cytometry (untreated cells)
- Treat second aliquot of cells with 0.1% Triton in PBS for 5 min on ice
- Analyze treated cells via flow cytometry
- Calculate FCDR index: (Fluorescence – autofluorescence of treated cells) / (Fluorescence – autofluorescence of untreated cells)