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Figure 1. Recombinant HEK293 Human 5-HTR2A  stably expressing 5-HTR2A

The recombinant HEK293 human 5-HTR2A cell line was validated for stable surface expression by flow cytometry. Staining with an anti-5-HT2A antibody produced a fluorescence median of 2693 for 5-HTR2A HEK293(C68), compared with 589 for parental HEK293 control, confirming stable expression of human 5-HTR2A receptor on cell membrane.

Figure 2. HTRF IP-One Assay with HEK293 Human 5-HTR2A (C68)

The 5-HTR2A HEK293(C68) cell line was functionally verified via HTRF IP-One assay. Serotonin triggers dose-dependent IP1 accumulation through Gq-coupled 5-HTR2A signaling, with EC50 = 10.84 nM. The parental HEK293 cells showed no response (EC50＞10000 nM), demonstrating specific receptor activity, suitable for high-throughput screening and batch QC testing.

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed HEK293 Human 5-HTR2A Cell Line complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.