SEAP Reporter Assay
| Materials | |
|---|---|
| Eppi centrifuge at RT
Heat block @65°C Substrate buffer @+4°C pNPP powder Calf Intestine Alkaline Phosphatase (CIAP) [stock: 1μL/mL, 1:1000 dilution) Multichannel pipette and plastic box for solution Spectrophotometer (VarioSkant) | |
| Assay buffer (100ml) | 100 mM Glycin pH 10.4 [10 M] ▶ 1 mL
1 mM ZnCl2 [1 M] ▶ 400 μL 1 mM MgCl2 [1 M] ▶ 400 μL Adjust pH with NaOH to pH 10.4 |
Procedure
- Transfer 1.3 ml of the conditioned cell culture medium (culture supernatant) at the required time point into an Eppendorf cup
- Spin for 3 min @800 rpm and transfer the supernatant in a new tube. Resuspend pelleted cells and put back in the corresponding dish.
- Spin for 5 min @13000 rpm and aliquote 500μL
- Incubate one aliquot for 30 min @65°C. In meantime, prepare substrate (1mg of pNPP in 10mL of Substrate buffer) and allow to equilibrate @RT
- Add 100μL/well of each sample in triplicate as well as positive (CIAP) and negative (Culture medium) controls.
- Dispense 100μL/well of substrate (+Buffer control, except Empty well control)
- Read absorbance @405nm reference 630nm