Hi Chloe, the detailed protocol is listed below:

1. Cell culture

1) Cells were seeded at a density of 1×10^5 cells per well in a 24 well plate, with a volume of 500 μL per well;

2) Prepare the protein using DMEM medium, discard the supernatant after cell adhesion, and add 500 μL of complete medium containing different concentrations of HMGB1 protein to a 24 well plate, so that the final concentration of HMGB1 protein in each well is 100, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, and 0 ng/mL;

3) Cells were cultured in a 5% CO2 incubator at 37 ℃ for 96 hours;

4) After reaching the cultivation time, discard the supernatant and wash it once with PBS for RNA extraction for qPCR detection.

2. RT qPCR detection of TNF-α secretion

2.1 RNA Extraction

1) After adding 1mL Trizol to the cells, let them stand at room temperature for 10 minutes, blow and mix well to fully lyse them.

2) Add 200 μL Chloroform/mL Trizol to Chloroform, shake well, and let it stand at room temperature for 3 minutes.

3) Centrifuge at 12000 rpm for 10 minutes, extract the upper aqueous phase and transfer it to another centrifuge tube.

4) Add equal volume of Isopropanol and mix well. Let it stand at room temperature for 20 minutes.

5) Centrifuge at 12000 rpm for 10 minutes and discard the supernatant.

6) Add 1mL of 75% Ethanol, gently shake the centrifuge tube, and suspend the precipitate.

7) Centrifuge at 12000 rpm for 5 minutes and discard the supernatant.

8) Dry at room temperature or vacuum dry for 5-10 minutes.

9) Add 50 μL of high-pressure sterilized DEPC H2O to dissolve the RNA sample.

10) Measure RNA concentration.

2.2 RT qPCR detection

The answer to this question is provided by MedChemExpress Technical Support.

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Here’s a very short, simple answer — concepts only (no wet-lab step-by-step):

• Goal: find HMGB1 concentration that gives 50% of the maximal TNF-α response (ED₅₀).
• Design: test a wide range of concentrations (serial dilutions), include negative/positive controls and replicates, measure TNF-α with a validated assay at one biologically relevant timepoint, and control for endotoxin/LPS.
• Analysis: fit a sigmoid (4-parameter logistic) to response vs. concentration and read ED₅₀ from the fitted curve; report ED₅₀ ± confidence intervals and show raw points + fit.
• Quick Python idea (replace concs and responses with your data):