MEGF10 Protein, Human (HEK293, His)

Materials
MEGF10 Protein, Human (HEK293, His) (HY-P76491)
Cell Line for Activity Assay: C2C12 Cells
Cell medium: DMEM +10% FBS+1% P/S

Penicillin-Streptomycin (100×), Sterile (HY-K1006)
Cell Counting Kit-8 (CCK8 Solution, HY-K0301)

Procedure
1. Using DMEM complete medium, seed cells into a 96-well plate at a density of 1500 cells/well, with a volume of 100 μL per well (Do not add cells to the peripheral wells of the 96-well plate; instead, add an equal volume of PBS solution).
2. After the cells adhere to the plate, discard the medium and subject the cells to starvation treatment overnight using DMEM medium containing 1% FBS.
3. Discard the medium in the wells, add 100 μL of DMEM medium (with 1% FBS) containing MEGF10 protein at different concentrations to the 96-well plate, making the final concentrations of PHPT1 protein in each well 0, 0.001, 0.01, 0.1, 1, 5, 10, 50, 100, 500, 2500, 5000, 10000, 25000 ng/mL.
4. Incubate the cells in a 37°C, 5% CO₂ incubator for 48 h.
5. After the incubation period, under dark conditions, add 10 μL of CCK8 solution to each well and incubate in a 37°C, 5% CO₂ incubator for 2 h.
6. Measure the OD value at 450 nm using a microplate reader.
7. Use GraphPad Prism software to plot a curve with the OD value as the ordinate and protein concentration as the abscissa, and calculate the ED₅₀ value.