Wnt-3a and R-spo1 conditioned media reporter assay
Natalia Martagón, Gurdrun Kliem
Abstract
Protocol designed to measure the activity of Wnt-3a or R-spondin-1 (Rspo1) conditioned media.
A reporter HEK cell line expressing luciferase under Wnt-3a stimulation is cultured with conditioned media followed by cell lysis and a luciferase reporter assay. Activity is compared to previous media batches or references.
Before start
Be sure to have access to:
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Luminometer with or without injection system.
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96 well plates appropriate for luminescence signal measurements
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Reagents for culturing reporter cell line. See reference (1)
*reagents might take a while to gather and prepare but once prepared they last for a long time
*time to produce conditioned media around 3 weeks. See reference protocols (2), (3).
Steps
Day 1: Seeding of Hek 293 STF cells
Culture HEK 293 STF CRL-3249™ (from now own referred as HEK-STF) according to the company specifications (1) until confluent and not too many passages old.
Seed 3 wells of HEK-STF cells per sample and following controls:
-negative control: conditioned media from L-cells (not transfected with luciferase construct) (4)
-positive control: previous batch with known activity, HEK-STF cells with recombinant Wnt-3a or agonist stimulation
-control lysates as blank for luminescence: HEK-SFT cells with HEK-medium only
Start with one almost confluent T75 culture bottle of HEK-STF cells
1x wash with DPBS: take out medium, add 5 mL DPBS, turn gently the bottle, take out DPBS
Detach cells with 1-2 mL Trypsin/EDTA (37°C ) and transfer to a conical tube with 8-9 mL HEK-medium
Centrifuge at 100-200 g for 0h 5m 0s
count cells (Neubauer chamber or automatic cell counter)
count cells (Newbauer chamber or automatic cell counter)
Seed cells in a 24 well plate. HEK-STF :
24 well Platte (0.05x106 cells/well). 1,3x106 cells / 13ml for the whole plate
cover each well with 0.5mL of HEK medium.
Day 2: cell stimulation
Aspirate medium and discard.
Add a total of 500µL of HEK medium with the following amount of conditioned medium (CM) to test:
- For R-spondin CM:
12.5% volumeWnt-3a CM +2.5% volumeRspo1 CM (3) - For Wnt3a CM .
50% volume
Incubate at stadard culture conditions for aprox. 24h 0m 0s
Day3 : luicferasse reaction
Luiferase solution
Prepare on the day luciferase solution. Keep in the fridge until ready to use
When using a plate reader with automatic injection, calculate the extra volume needed for it (ca 3 mL)
fill to final volume with Assay reagent solution
| A | B | C | D |
|---|---|---|---|
| Reagent | units | [stock] | [working] |
| DTT | M | 1 | 0,033 |
| CoA | mM | 10 | 0,266 |
| Luciferin | mM | 20 | 0,467 |
| ATP | mM | 100 | 0,633 |
Prepare 1x Lysis medium from the stock with destiled water
Cell lysis
Aspirate medium from HEK-STF cells
Add 150µL /well of 1x Lysis buffer.
Leave the plate for 0h 20m 0s at Room temperature. (Note: pippeting seems to make cell aggregates and bubbles)
Check under the microscope that most cells are lysed
Shake gently for 0h 5m 0s on plate shaker
Transfer 20µL of each sample to a 96 well plate for luminescence read.
Avoid bubbles or cell clumps. Recommended pipetting scheme as in the 24 well plate of HEK-STF culture-
If using a plate reader without automatic injection:
Take to the plate reader with luci. solution, multichanel pipette, and reagend reservoir
Once the plate reader is set, add 100µL of luciferse solution per well and read.
For accuracy, use a multichanel pipette and add the solution covering not one sample at a time but one of the triplcates of all samples at one time.
(signal decreases ca. 20% in the first 10 minutes)
Check that the relative luminescence units from negative controls are orders of magnitude lower that test samples. Average values from triplicates.
Values below more than 50-60% lower than a working batch of conditioned media are considered of poor quality.
Examples of equipment and methods: Berthold Tristar-GAS ISRE Luciferase Assay, SpectraMax i3x-SpectraMax Glo Steady-Luc Reporter Assay
