B220 microbeads

Spleen cells derived from BALB/c mice were harvested and their red blood cells were lysed. B cells in splenocytes were first depleted using B220 microbeads (Miltenyi Biotec) via negative selection. Cells (1 × 10⁶/sample) then were stained with diluted serum (1/10) from naïve or transplanted C57BL/6 mice. They were further incubated with PE-anti-mouse IgM or FITC-anti-mouse IgG (Biolegend). The mean fluorescence intensity was determined by a flow cytometer (FACSCalibur, BD Biosciences).

B220⁺ B cells were sorted from splenocytes of 7- to 9-week female Balb/c, Stch^f/f, and CD19^creStch^f/f mice (3 mice per group) using B220 microbeads (Cat No. 130–049-501, Miltenyi Biotec, Germany), B220⁺ B cells were stimulated with 10 μg/ml LPS (L2630, Sigma, St Louis, MO) for 3 days in vitro as previously described [18 (link)]. SP 2/0 cells (ATCC® CRL-1581, Rockville, MD, USA) were thawed, passaged three times, and then cultured for 2 days in fresh medium. RNeasy Mini Kit (Qiagen, Venlo, Netherlands) was used to isolate and purify total RNA from cells. NanoDrop®ND-1000 spectrophotometer and Agilent 2100 Bioanalyzer and RNA 6000 NanoChips (Agilent, Palo Alto, CA, USA) were used to determine RNA concentration and quality, respectively. TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina) was used to prepare Libraries. Transcripts were analyzed by RNA-sequencing (Genewiz Corp., Suzhou, China) using a standard method [18 (link)].

MBCs were generated by immunization of congenic donor mice (Ly5.1 or IgHa). At least 8 weeks after immunization donor mice were sacrificed and spleens isolated in RPMI media containing 2% FCS and antibiotics. A single cell suspension of the spleens was prepared and red blood cells were lysed using ACK buffer (0.15 M ammonium chloride, 0.01 M potassium hydrogen carbonate, pH 7.2–7.4). The splenocytes were PNA⁻ and B220⁺ MACS purified. For PNA negative purification splenocytes were labeled using PNA-biotin (Vector Labs, B-1075) and PNA⁺ cells were depleted by Strepravidin MicroBeads (Milteny Biotec, 130-048-101) according to the manufacturer's protocol. Positive selection using B220 MicroBeads (Milteny Biotec, 130-049-501) was performed according to the manufacturer's protocol.
Purified cells from 1/3 of a donor spleen (Ly5.1 or IgHa; ~1–3 × 10⁶ cells) were adoptively transferred i.v. into congenic host mice (Ly5.2 or IgHb). Control mice received PNA⁻ and B220⁺ purified splenocytes from naïve congenic mice. One day after MBC transfer host mice were challenged with 50 μg Qβ VLPs i.v.

B cells were extracted as described. Spleens from C57/Bl6 wild-type or Trp53^−/− mice were collected four hours after exposure to 7 Gy whole-body irradiation and from a control cohort of mice. Single-cell suspensions were obtained by pressing the spleens through nylon cell strainers and subsequent hypotonic lysis of red blood cells. To isolate B cells, we incubated single-cell suspensions with B220 MicroBeads (Miltenyi Biotech) and enriched them by magnetic cell sorting (MACS), according to the manufacturer instructions (Miltenyi Biotech). The column flow through was kept to represent the non-B cell population.

Single-cell suspensions were prepared from spleens of the indicated mice. For sorting ABCs, splenocytes were pre-enriched for B cells with B220 microbeads (Miltenyi Biotec; #130-049-501) following manufacturer’s instructions. B cells were stained with CD11c-APC (N418; 1:400), CD11b-FITC (M1/70; 1:400), CD19-PB (HIB19; 1:400), B220-APC/Cy7 or B220-PE/Cy7s (RA3-6B2; 1:400), and CD23-PE (B3B4; 1:200) and were sorted on FACS Aria or Influx (BD). ABCs were collected for RNA or cultured for 7d with 1μM Imiquimod in RPMI 1640 medium (Corning) supplemented with 10X FBS (Atlanta Biologicals), 100 U/mL Penicillin (Corning), 100 mg/mL Streptomycin (Corning), 1X non-essential amino acids (Corning), 2mM l-Glutamine (Corning), 25 mM HEPES (pH7.2-7.6; Corning), and 50 μM β-Mercaptoethanol. For sorting CD11c⁺ and CD11c⁻ GC B-like cells and PB/PCs, splenocytes were pre-enriched using biotinylated antibodies against B220 and CD138 and with streptavidin-conjugated microbeads (Miltenyi Biotec; #130-048-101) following manufacturer’s instructions. Cells were stained with CD11c-APC/Cy7 (N418; 1:400), CD19-PB (HIB19; 1:400), CD138-APC (281-2; 1:600), CD38-PE/Cy7 (90; 1:600), GL7-FITC (1:600), and TACI-PE (8F10; 1:400) and were sorted on FACS Aria or Influx (BD).

B-cell purification and in vitro differentiation were previously described (29 (link), 30 (link)). Briefly, splenic B220⁺ B cells were separated by B220 microbeads (Cat No. 130-049-501, Miltenyi Biotec). B cells were stimulated with 10 μg/ml LPS (Sigma L2630 from Escherichia coli 0111:B4; Sigma, St Louis, MO) in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, penicillin (100 IU/ml), streptomycin (100 μg/ml), and 50 mM 2-mercaptoethanol.