CRELD2 Protein, Mouse (HEK293, His)

Cell Culture
Cell line used for viability testing: ATDC5 cells
Cell culture medium: DMEM medium + 10% FBS + 1% P/S
Test protein: CRELD2 Protein, Mouse (HEK293, His) (HY-P76289)
Penicillin-Streptomycin (100X) (HY-K1006)
Cell Counting Kit-8 (HY-K0301)

Procedures
1. Dilute different concentrations of Mouse CRELD2 protein with PBS and add to a 96-well plate at 40 μL per well, so that the final concentration of CRELD2 protein in each well is 0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 2.5, and 5 g/mL.
2. The 96-well plate was placed in a ventilated dry place for 1h and then placed at 4℃ overnight.
3. Discard the protein and wash the plate with serum-free DMEM twice at 200 μL each time.
4. Using DMEM complete medium, inoculate the cells at a density of 5×10⁴ cells/well in a coated 96-well plate with a volume of 100 μL/well.
5. Cells were incubated at 37°C, in a 5% CO₂ incubator for 1 h.
6. The medium in the wells was discarded and washed twice with PBS.
7. Under the condition of light-avoidance, 100 μL of 10% CCK8 solution was added to each well, and the cells were incubated at 37°C, 5% CO₂ incubator for 2h.
8. The optical density (OD) value at 450 nm was measured using a microplate reader.
9. Using GraphPad Prism software, plot a curve with OD values on the y-axis and protein concentration on the x-axis to calculate the ED₅₀ value.