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

2025-02-27T12:41:55Z

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

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{| class="wikitable"
|+
!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

SEAP Reporter Assay - Revision history