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. CD25 deficiency results in a syndrome similar to IPEX with severe enteropathy, diabetes mellitus, autoimmune hemolytic anemia, eczema, and lymphoproliferation. Importantly, patients with CD25 deficiency exhibit several unique features not seen in IPEX, namely chronic herpetic viral infections and an increased susceptibility to infections.
Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.
Cell Line Name:
Ba/F3 STAT5-Luc2-IL2RABG Cell Line
Host Cell Line:
Mouse Ba/F3 cell line
Stable Ba/F3 IL2RABG cell line expressing exogenous luciferase gene under the control of STAT5 responsive element.
One vial of frozen cells (5X106 per vial)
Stable in culture over a minimum of 10 passages
Drug screening and biological assays
70% RPMI1640+20% FBS+10% DMSO
RPMI1640+10%FBS+1μg/mL Puromycin +1mg/mL Hygromycin B
Mostly single, round (some polymorph) cells in suspension
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 20 hours
Cell Line Generation
Ba/F3-STAT5-Luc2-IL2RABG Cell Line was generated using a lentiviral vector expressing the human IL2RABG sequence.
Characterization using PCR sequencing and WB
1. Prewarm culture medium (RPMI1640 + 10% FBS + 1μg/mL Puromycin + 1mg/mL Hygromycin B) 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.
1. Prepare the freezing medium (70% RPMI-1640 + 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
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.
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