Class switch recombination assays were performed essentially as previously described (Noordermeer et al, 2018 (link)). 1 × 105 CH12F3‐2 cells were plated in 24‐well plates in growth medium supplemented with 1 μg/ml anti‐CD40 antibody (eBioscience), 1 ng/ml TGF‐β (R&D Systems), and 10 ng/ml mIL‐4 (R&D Systems). After 48 h, cells were harvested, stained with anti‐IgA‐PE (Southern Biotech), and fixed with 4% formaldehyde. Fluorescence signal was acquired on an Attune NxT Flow Cytometer (Thermo‐Fisher). Data were analyzed using FlowJo software (BD Biosciences).
Protocol detail
Measurement of Class Switch Recombination
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Anti antibody
Anti iga
Assays
Cells
Class switch recombination
Fluorescence
Formaldehyde
Growth medium
Tgf β
Corresponding Organization :
Other organizations : Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto
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Variable analysis
independent variables
- Anti-CD40 antibody (1 μg/ml)
- TGF-β (1 ng/ml)
- MIL-4 (10 ng/ml)
- Class switch recombination efficiency (measured by anti-IgA-PE staining and flow cytometry)
- CH12F3-2 cell line
- Growth medium
- Incubation time (48 h)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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