ESO wikiadmin: Created page with "{| class="wikitable" |+ !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..."

2025-02-27T12:45:32Z

Created page with "{| class="wikitable" |+ !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..."

New page

{| class="wikitable"
|+
!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)

Measurement of lipid raft localization via flow cytometry - Revision history