Click it plus edu alexa fluor 647 kit

1 mg of EdU (dissolved in water) was injected intraperitoneally into mice 1 h before euthanasia. Mice were perfused with 5% NBF and the meninges were removed as described above. EdU was detected using a Click-iT Plus EdU Alexa Fluor 647 kit (ThermoFisher) and was imaged with an Olympus FV1200 laser scanning confocal microscope. Quantification of EdU⁺ cells was performed by histo-cytometry as described below.

B cell development in bone marrow and spleen of sex-matched Fam72a^−/− and Fam72a^+/+ littermates (6–8 weeks old) was evaluated (Supplementary Fig. 2) as described^{33 (link)}. To induce ex vivo CSR to different immunoglobulin isotypes, splenic B cells were purified with a mouse B cell isolation kit (StemCell Technologies), and then stimulated with LPS (Sigma-Aldrich) in combination with various cytokines, as previously described^{33 (link)}. Cells were collected at day 4 after stimulation and were stained with antibodies against mouse IgG1 (PE, BD Pharmigen, cat. no. 550083; 1: 150 dilution), IgG2b (PE, SouthernBiotech, cat. no. 1090–09S; 1:150), IgG3 (FITC, BD Pharmigen, cat. no. 553403; 1:100), IgE (FITC, BD Pharmigen, cat. no. 553415; 1:100) or IgA (PE, SouthernBiotech, cat. no. 1040–09; 1:150) to assess CSR. For the cell cycle analysis, splenic B cells were stimulated with LPS for 2.5 days and analysed with the Click-iT Plus EdU Alexa Fluor 647 kit (ThermoFisher Scientific) as described^{33 (link)}. IgG1 CSR was induced in carboxyfluorescin diacetate succinimidyl ester (CFSE)-pulsed splenic B cells as we previously described^{33 (link)}. The apoptosis of LPS stimulated splenic B cells was assayed using an APC-conjugated Annexin V Apoptosis Detection Kit (eBioscience). The flow cytometric data were analysed with a FlowJo X10 software.

For proliferation assay, BMDEs of culture day 8 were seeded at 0.5×10⁶ cells/mL in 500 μL BM-medium in a 48 well plate and stimulated with 5 μg/mL rat IgG2a, к isotype control antibody or purified rat anti-mouse Siglec-F antibody (clone E50–2440) and optional 10 ng/mL recombinant murine IL-33 (R&D Systems) for 24 hrs. Additionally, 10 μM EdU (Invitrogen) per well were added and cells were incubated for further 24 hrs at 37°C 5% CO2. Then cells were stained for surface antigens followed by EdU staining with the Click-iT Plus EdU Alexa Fluor 647 Kit (Invitrogen) according to manufacturer’s instructions. For apoptosis assay, mature BMDEs were seeded at 0.2×10⁶ cells/mL in 200 μL BM-medium in a 96 well plate and stimulated as in the proliferation assay described above for 24 hrs. For flow cytometry staining, Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl₂) was used instead of FACS buffer. AnnexinV in FITC or APC (ImmunoTools) was used and DAPI (Sigma/Merck) was added to each sample directly before measuring in the flow cytometer.