In vitro GC (iGC) culture was performed as described.44 (link) B cells were isolated from indicated mice using a negative selection B cell isolation Kit (StemCell Technologies). B cells were overlaid on irradiated (120gy) 40LB fibroblasts in RPMI (Corning) containing 10% Hyclone Fetal Bovine Serum (GE Life Sciences), Penicillin Streptomycin (Corning), Glutamax (Gibco), Sodium Pyruvate (Gibco), 5μM 2-Mercaptoethanol, and HEPES Buffer (Corning) supplemented with 5 ng/mL IL-4 (Peprotech) at 37C in 5% CO2. On day 4 of the culture GC B cells were purified from the fibroblasts by negative selection using biotinylated H-2Kd (SF1–1.1; Biolegend) and CD138 (281–2; BD Biosciences) Abs and stimulated with various concentrations of IL-21 (Peprotech). Cells were harvested 12- and 24-h post-stimulation and processed for RT-PCR (described below) and flow cytometry (described above).
Negative selection b cell isolation kit
Manufactured by STEMCELL
The Negative Selection B Cell Isolation Kit is a laboratory product designed to isolate B cells from a mixed cell population. It utilizes an antibody-based negative selection approach to deplete non-B cells, allowing for the enrichment of the desired B cell population. The kit provides the necessary reagents and protocols to perform this isolation process.
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Lab products found in correlation
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Cd138 281 2, by BD (1 mentions)
Hyclone fetal bovine serum, by GE Healthcare (1 mentions)
Il 21, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Roswell park memorial institute (rpmi), by Corning (1 mentions)
Hepes buffer, by Corning (1 mentions)
Anti mouse cd38, by BioLegend (1 mentions)
Zombie yellow viability stain, by BioLegend (1 mentions)
Trustain fcx antibody, by BioLegend (1 mentions)
2 protocols using negative selection b cell isolation kit
1
In Vitro GC B Cell Culture
2
Isolation and Sorting of Germinal Center B Cells
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GC B cells were generated and sorted using a previously described protocol (Hwang et al., 2015 (link)). Briefly, 6–12-week-old M2A-GFP KI mice were immunized with sheep’s red blood cells. After 8–10 days, total B cells from the spleens and lymph nodes were isolated using the Negative Selection B cell isolation kit (StemCell Technologies) according to the manufacturer’s instructions. Dead cells were stained using Zombie Yellow viability stain (BioLegend) and Fc receptors were blocked with the mouse TruStain FcX antibody (#156604). Cells were immunostained with anti-mouse CD38 (#102719), B220 (#103235), and GL-7 (#144617) purchased from BioLegend. GC B cells were sorted on a BD Aria III FACs sorter (Beckton Dickinson) for GFP+, Zombie Yellow-, B220+, CD38low and GL-7+ cells, and were used immediately.
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