TITLE: Wnt-3a and R-spo1 conditioned media reporter assay
AUTHORS: Natalia Martagón, Gurdrun Kliem

[DESCRIPTION]
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:

- Luminometer with or without injection system.
- 96 well plates appropriate for luminescence signal measurements 
- 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]
SECTION: Day 2: cell stimulation
2. Add 250 µL  of conditioned medium (CM) to test in the desired concentration
R-spondin: 12.5 % volume  Wnt-3a CM + 2.5 % volume  Rspo1 CM (3)
Wnt3a CM .50 % volume  250 µL Sample + 250 µL HEK medium



SECTION: Day 1: Seeding of Hek 293 STF cells
1.1. Start with one almost confluent T75 culture bottle of HEK-STF cells
SECTION: Day 1: Seeding of Hek 293 STF cells
1.2. 1x wash with DPBS: take out medium, add 5 mL DPBS, turn gently the bottle, take out DPBS

SECTION: Day 1: Seeding of Hek 293 STF cells
1.3. Detach cells with 1-2 mL Trypsin/EDTA (37 °C ) and transfer to a conical tube with 8-9 mL HEK-medium

SECTION: Day 1: Seeding of Hek 293 STF cells
1.4. Centrifuge at 100-200 g for 5 min
 count cells (Neubauer chamber or automatic cell counter)
SECTION: Day 1: Seeding of Hek 293 STF cells
1.5. count cells (Newbauer chamber or automatic cell counter)

SECTION: Day 1: Seeding of Hek 293 STF cells
1.6. 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  0.5 mL of HEK medium.

SECTION: Day 1: Seeding of Hek 293 STF cells
1. 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


SECTION: Day 2: cell stimulation
2.1. Incubate at stadard culture conditions for aprox. 1440 min 

SECTION: Day3 : luicferasse reaction
3. 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


 

SECTION: Day3 : luicferasse reaction
5. Cell lysis
Aspirate medium from HEK-STF cells
SECTION: Day3 : luicferasse reaction
5.1. Add 150 µL /well of  1x Lysis buffer. 

SECTION: Day3 : luicferasse reaction
6. Leave the plate for 20 min at Room temperature. (Note: pippeting seems to make cell aggregates and bubbles)

SECTION: Day3 : luicferasse reaction
7. Check under the microscope that most cells are lysed
SECTION: Day3 : luicferasse reaction
8. Shake gently for 5 min on plate shaker

SECTION: Day3 : luicferasse reaction
9. Transfer 20 µL  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-

SECTION: Day3 : luicferasse reaction
10. 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 100 µL  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.
SECTION: Day3 : luicferasse reaction
11. 
(signal decreases ca. 20% in the first 10 minutes)
SECTION: Day3 : luicferasse reaction
12. 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
SECTION: Day3 : luicferasse reaction
4. Prepare 1x Lysis medium from the stock with destiled water