View source for Measurement of lipid raft localization via flow cytometry

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!Materials
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|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
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=== Procedure ===
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* 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)