Cell Adherence assay

Materials

Materials
	96 well plates; 15 ml und 30 ml Falcon-tubes
	coating solution for wells e.g. collagen type 1 (25 µg/ml)
	Soybean Trypsin/Inhibitor (12.5 mg/50 ml) in Suspensionen medium- sterile
	Crystal violet (250 mg/ 5 ml 96% Ethanol stock solution)
	Suspension medium (DMEM +0,25%BSA) sterile
	diluted Crystal violet 1:100 in 0.1M Borat buffer pH: 9.2 (freshly prepared!)
	4% PFA (Paraformaldehyde)
	1 x PBS, pH 7.4
	ELISA Microplate Reader
	Acetic acid 10 mM; 1mg/ml BSA
	Starvation medium: DMEM + 0.25% BSA

Assay

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• transfected and / or infected cells are serum starved one night before experiment
• cells are washed two times with PBS, then treated with 1 ml Trypsin/EDTA per plate for about 2 min at 37°C - Trypsin activity is then stopped by adding 5 ml soybean-Inhibitor followed by centrifugation: 600 rpm, 3 min
• Discard supernatant and resuspend cells in 10 ml Suspension medium, cells are shaken carefully for 1 h at 37°C (use 50 ml Falkon Tubes )
• In the meantime calculate the number of cells necessary for the experiment (5 x 104 cells per 96 well) in 100 - 120µl medium - coat wells before.
• To coat the wells: 100 µl 1 x PBS with the appropriate substrate (e.g. collagen 25µg/ml) over night at 4°C or 1h at 37°C.
• Next day replace the substrate solution with 0.2 % BSA to block the wells for additionally 1 h at 37°C.
• suck off BSA, seed calculated number of cells into the wells and incubate cells for another 1h at 37C°. (Time of adhesion can vary from cell type to cell type)
• Analysis of adherend cells: after incubation time wells are washed 3 times with 120 µl PBS/Mg/Ca (small pipette and equal pressure) and afterwards fix remaining cells with 100 - 120 µl 4% PFA per well for 25 min at RT or 4°C over night.
• Next day cells are washed at least 3 to 4 times with PBS, dried shortly and stained with diluted crystal violet - 120µl per well, 1h with gentle shaking
• take away the crystal violet and wash again 3 to 4 times each well with H₂O or PBS - cells are shortly dried and analysed using a Mikroplate Reader (Varioskan Flash) at a wave length of 550 nm
• in addition cells could be solved in acetic acid,10 mM;1 mg/ml BSA), 100µl per well for 1 h with gentle shaking and measure again - samples should be diluted 1/10.