KC-1978

Ba/F3-IL7Ra-CRLF2-Cell-Line

23165

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Background of Ba/F3-IL7Ra-CRLF2-Cell-Line

Interleukin-7 receptor alpha (IL7RA) is a receptor subunit with dual roles. Upon association with the interleukin-2 receptor gamma (IL2Rγ) subunit, it forms the Interleukin-7 (IL7) receptor and when bound to cytokine receptor-like factor 2 (CRLF2) subunit, it constitutes the thymic stromal lymphopoietin (TSLP) receptor. Loss-of-function mutations in IL7RA are associated with absent B cells and T cells in mice but with the absence of only T cells in humans.

Specifications

Catalog Number	KC-1978
Cell Line Name	Ba/F3-IL7Ra-CRLF2-Cell-Line
Clone Number	3#
Host Cell Line	Mouse Ba/F3 cell line
Description	Stable Ba/F3 clone expressing exogenous IL7Ra and CRLF2 genes.
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS+20ng/mL IL-7
Selection Marker	Puromycin
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Ba/F3-IL7Ra-CRLF2 cell Line was generated using retrovirus vector expressing human IL7Ra and CRLF2 sequence.

Characterization

Figure 1: Characterization of IL7Ra and CRLF2 in the Ba/F3 stable clone using PCR sequencing.

Figure 2: Characterization of IL7Ra overexpression in the Ba/F3 stable clone using FACS.

Figure 3: Ba/F3-IL7Ra-CRLF2 cell line was seeded into the 96-well plate, and treated with TSLP at a maximum concentration of 1μg/mL for 72 hours, then readout with CTG system.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS+20ng/mL IL-7)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Scott LM. Lymphoid malignancies: Another face to the Janus kinases. Blood Rev. 2013 Mar;27(2):63-70. doi: 10.1016/j.blre.2012.12.004. Epub 2013 Jan 20. PMID: 23340138.
Geron I, Savino AM, Fishman H, Tal N, Brown J, Turati VA, James C, Sarno J, Hameiri-Grossman M, Lee YN, Rein A, Maniriho H, Birger Y, Zemlyansky A, Muler I, Davis KL, Marcu-Malina V, Mattson N, Parnas O, Wagener R, Fischer U, Barata JT, Jamieson CHM, Müschen M, Chen CW, Borkhardt A, Kirsch IR, Nagler A, Enver T, Izraeli S. An instructive role for Interleukin-7 receptor α in the development of human B-cell precursor leukemia. Nat Commun. 2022 Feb 3;13(1):659. doi: 10.1038/s41467-022-28218-7. PMID: 35115489; PMCID: PMC8814001.