Goat f ab 2 anti human igm unlb

Naïve, memory and GC B cells were incubated at a density of 10⁶ cells/ml with or without an activation cocktail in IMDM medium supplemented with 10% FBS, 1% Penicillin/Streptomycin, and 1% glutamine at 37 °C for various time periods prior to analysis by flow cytometry to determine total protein levels, by iFACS to calculate DM_free/CLIP_freq, or by 3D-SIM to quantify protein co-localization. The activation cocktail contained 20 ng/ml of IL-4 (Peprotech), 20 ng/ml of IL-2 (BioLegend), 200 ng/ml of MEGACD40L (human CD4 ligand, Enzo Biochem Inc), 5 μg/ml of Goat F(ab’)2 anti-human IgG-UNLB mouse adsorbed and 5 μg/ml Goat F(ab’)2 anti-human IgM-UNLB (Southern Biotech).

Tonsil-derived cells (5x10⁵/200 μl) were plated in medium alone (RPMI supplemented with 10% fetal calf serum, 2 mM l-glutamine, 10 mM Hepes, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 1× nonessential amino acids), or in medium plus 10 μg/ml CpG (Primm), 10 μg/ml goat F(ab')2 anti-human IgM-UNLB (SouthernBiotech), or Staphylococcus aureus (S. aureus) Cowan I (SAC, Calbiochem) at the final dilution of 1:20000. The incubation was performed in the presence of 1000 U/ml IL-2 (Peprotech) for 2 days at 37°C with 5% CO₂. Brefeldin-A (Sigma) was added at 5 μg/ml 3 h before fixation and staining. Alternatively, 1.5 x 10⁶ cells/200μl RPMI supplemented with 10% fetal calf serum were plated in a 96-well plate and immediately stained using the antibodies listed above.