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Figure 1. Recombinant CX3CR1/CHO-K1 stably expressing CX3CR1

Blue curve: CX3CR1 / CHO-K1 cells stained with anti-CX3CR1 antibody, mean FL4-A = 4.46E5. Red curve: wild-type CHO-K1 negative control stained with anti-CX3CR1 antibody, mean FL4-A = 932. The data verifies high CX3CR1 expression on the recombinant cell membrane.

Figure 2. HTRF CAMP Assay With CX3CR1 CHO-K1 Cells (C15)

HTRF Ratio was measured to reflect intracellular cAMP level. IC50 = 2.53 nM. The curve demonstrates that CX3CL1 stimulation inhibits cAMP production via CX3CR1 (Gi-coupled receptor), confirming robust receptor functional activity.

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 CHO-K1 Human CX3CR1 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.