KC-2472

BaF3-STAT5-Luc2-IL2RBG Cell Line

24128

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Background of BaF3-STAT5-Luc2-IL2RBG Cell Line

IL-2R is made up of three chains, α (CD25), β (CD122), and γ (CD132), of which only the α-chain is specific for IL-2. Signaling takes place mainly via CD122. Activated T cells express the high affinity receptor composed of all three chains. However, cells other than T cells, such as monocytes, express functional CD122 and CD132 and have the ability to signal following ligation of the intermediate affinity receptor consisting of the heterodimer formed by CD122 and CD132. CD25 (α chain of the high-affinity IL-2 receptor) deficiency is an autosomal recessive disorder due to mutations in the IL2RA gene. T regulatory cells constitutively express CD25 and respond to IL-2 generated by T cells during an immune response for their immunoregulatory functions.

Specifications

Catalog Number	KC-2472
Cell Line Name	BaF3-STAT5-Luc2-IL2RBG Cell Line
Host Cell Line	BaF3-STAT5-Luc2
Description	Stable BaF3 cell line expressing exogenous luciferase gene under the control of IL2RBG signaling pathway
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% RPMI640+20% FBS+10% DMSO
Propagation Medium	RPMI1640 + 10% FBS+8ng/ml mIL-3+1mg/ml Hygromycin B+1μg/ml Puromycin
Selection Marker	Puromycin, Hygromycin B
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 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

BaF3-STAT5-Luc2-IL2RBG cell line was generated using lentivirus expressing IL2RBG sequence.

Characterization

Figure 1. Characterization of human CD122 overexpression in Ba/F3 stable clones using FCAS.

Figure 2. Characterization of human CD132 overexpression in Ba/F3 stable clones using FCAS.

Figure 3. Starved Ba/F3-STAT5-Luc2-IL2RBG cell line was seed into the 96-well plate, and treated with serial diluted human IL-2 for 6 hours, then readout with Bright-lite™ Luciferase Assay system.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS, 8ng/ml mIL-3,1mg/ml Hygromycin and 1μg/ml Puromycin)in a 37°C water bath. 2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes. 3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol. 4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium. 5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet. 6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask. 7. Incubate the flask at 37°C, 5% CO₂ incubator. 8. Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use. 2. Keep the freezing medium on ice and label cryovials. 3. Transfer cells to a sterile, conical centrifuge tube, and count the cells. 4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium. 5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium. 6. Aliquot 1 mL of the cell suspension into each cryovial. 7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer. 8. Transfer vials to liquid nitrogen for long-term storage

References

1.James W. Verbsky, John R. Routes, Infections, Immune Disorders, and Autoinflammatory Diseases. Nelson Pediatric Symptom-Based Diagnosis, 2018. 2.MATEEN ARASTU, SHAZIA KHAN, CHANDRABALA SHAH, et al. Functional IL-2 receptor #beta#(CD122) chains are expressed by fibroblast-like synoviocytes:activation by IL-2 stimulates monocyte chemoattactant protein-1 production[J]. The Journal of Immunology: Official Journal of the American Association of Immunologists,2001.