ESO wikiadmin: Created page with "{| class="wikitable" |+ !Materials ! ! ! |- |'''Wash solution''' |1x PBS mit .0.0 5% BSA | | |- |'''Block solution''' |2% BSA in 1 xPBS | | |- |'''Permeabilization solution''' |0.1% Triton X-100 (0.1% in PBS) | | |- |'''Buffer for antibody''' |0.1% BSA in x PBS | | |- |'''Reaction buffer''' |10 ml Lösung '''B''' + 0.5 ml Lösung '''A''' | | |- |'''Solution A''' |120 mg Tetramethylbenzidin in 5 ml Aceton + 45ml EtOH add 100µl H₂O₂ | | |- |'''Soluti..."

2025-02-27T11:43:05Z

Created page with "{| class="wikitable" |+ !Materials ! ! ! |- |'''Wash solution''' |1x PBS mit .0.0 5% BSA | | |- |'''Block solution''' |2% BSA in 1 xPBS | | |- |'''Permeabilization solution''' |0.1% Triton X-100 (0.1% in PBS) | | |- |'''Buffer for antibody''' |0.1% BSA in x PBS | | |- |'''Reaction buffer''' |10 ml Lösung '''B''' + 0.5 ml Lösung '''A''' | | |- |'''Solution A''' |120 mg Tetramethylbenzidin in 5 ml Aceton + 45ml EtOH add 100µl H2O2 | | |- |'''Soluti..."

New page

{| class="wikitable"
|+
!Materials
!
!
!
|-
|'''Wash solution'''
|1x PBS mit .0.0 5% BSA
|
|
|-
|'''Block solution'''
|2% BSA in 1 xPBS
|
|
|-
|'''Permeabilization solution'''
|0.1% Triton X-100 (0.1% in PBS)
|
|
|-
|'''Buffer for antibody'''
|0.1% BSA in x PBS
|
|
|-
|'''Reaction buffer'''
|10 ml Lösung '''B''' + 0.5 ml Lösung '''A'''
|
|
|-
|'''Solution A'''
|120 mg Tetramethylbenzidin in 5 ml Aceton + 45ml EtOH add 100µl H2O2
|
|
|-
|'''Solution B'''
|30 mM potassium citrate, pH 4.1
|
|
|-
|'''Starvation-medium'''
|DMEM +0.5% CS
|
|
|-
|'''Suspension medium'''
|DMEM + 0.25% BSA
|
|
|-
|'''Soybean-inhibitor'''
|0.5 mg/ml soybean trypsin in DMEM.
|
|
|}

=== Procedure ===
----

* Coat 96well-plates with e.g. collagen type1 or poly-Lysin over night
* serum starve cells (0.5% FCS) over night
* Next day cells are washed with 1x PBS, detached by limited trypsin/EDTA digestion, which is stopped by addition of soybean trypsin inhibitor
* pellet cells by centrifugation for 3min / 6000rpm (in the meantime cells are counted using the CASY)
* Detached cells are kept in suspension medium for 1 h at 37°C, and replated at 5 x 104 cells/well into wells coated with collagen.
* After 75 min cells were treated or not with 1M MnCl 1µl / well for about 5 min at 37°C and transferred to ice
* Cells are fixed with 4% paraformaldehyde in PBS for at least 30 min, washed with PBS and permeabilized with Triton X-100 for 15min
* cells are washed again 3 to 4 times with PBS and blocked with 2% BSA for 20 min
* cells are incubated with the mAbs 9EG7 (1:600) or AIIB2 (1:750) in blocking buffer to mark activated (9EG7) or total (AIIB2) β1 integrins.
* After incubation with the primary antibody, cells are washed with PBS, blocked 2% BSA for 20 min
* add secondary antibody (ProteinA-HRP or ProteinG-HRP) and incubate cells for 1h in the dark
* Wash and block cells for 20 min
* Start reaction: Cells are incubated with 100 μl / well of substrate solution (substrate solution is prepared by mixing 0.5 ml of TMB solution A with 10 ml solution B)
* The enzymatic colour reaction is stopped using 2 M H2SO4 (100μl/well) and the absorbance is determined at 450 nm.

Determination of Integrin Activity (ELISA-based) - Revision history