Anti cd43 microbeads

Mouse strains and cell lines used in the study are listed in key resource table. Mouse Embryonic Fibroblasts were produced at embryonic day 13.5 from timed breeding between 8-12 weeks old Pole4^+/−-Trp53^+/- males and females mice in C57BL/6 background. All animal experimentations were undertaken in compliance with UK Home Office legislation (project license number 70/8527) under the Animals (Scientific Procedures) Act 1986. Primary Pole4^+/+ and Pole4^-/- MEFs in a Trp53^{+/+ , +/-}and^-/- background were cultured at 37°C/ 5% CO₂/ 5% O₂ in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 15% fetal bovine serum (FBS) (Sigma) and 1% penicillin-streptomycin (Invitrogen). Primary splenocytes from 6-12 week old Pole4^+/+ and Pole4^-/- sex-matched male and female mice were purified as previously described (18). Resting B cells were isolated from wild-type and Pole4 KO mouse spleens with anti-CD43 MicroBeads (Miltenyi Biotech) and cultured in RPMI with 10% FBS. Cdkn1a^+/+ and ^-/- primary MEFs were kindly provided by Valery krizhanovsky, Weizmann Institute of Science, Israel.

WT and DKO MEFs, naive B cells, ESCs, and iPSCs were prepared in our lab. The detailed cell culture conditions were described in our previous publications [19 (link), 25 (link), 28 (link)]. MEFs were prepared from E13.5 embryos and maintained in DMEM medium plus 10% FBS and 1% Pen Strep. Mouse ESCs and iPSCs were maintained on MEF feeder cells in the DMEM medium, which contained 15% FBS, 1% Pen Strep, Glutamax, Sodium Pyruvate, MEM Non-Essential Amino Acids, and 0.1 mM β-mercaptoethanol. For feeder-free culture, mouse ESCs and iPSCs were maintained in a knockout DMEM medium with 20% KOSR (Thermo Fisher Scientific, Cat# 10828028), 1% Pen Strep, Glutamax, Sodium Pyruvate, MEM Non-Essential Amino Acids 0.1 mM, β-mercaptoethanol, 1000  U mL⁻¹, ESGRO^®-2i Supplement Kit (1000×), and 1000  U mL⁻¹ LIF (Millipore Sigma, ESG1121). Naive B cells were isolated from the spleens of 2-month-old WT and DKO male mice by immunomagnetic depletion, using anti-CD43 MicroBeads (Miltenyi Biotech). Purified B cells were cultured in RPMI 1640 media supplemented with 10% FBS and 1% Pen Strep, 1X GlutaMax™, 50 μM 2-mercaptoethanol.

Primary B cells were isolated from mouse spleens by immunomagnetic depletion with anti-CD43 MicroBeads (Miltenyi Biotech) [45] (link). The harvested cells were stimulated in the presence of 25 μg/mL LPS (Roche) and 50 ng/mL IL-4 (Sigma-Aldrich) for three days. After the stimulation, RNA was isolated for a further analysis.