Macs separation ls columns

B cells were isolated from single-cell splenocyte suspensions using B cell isolation kits (Miltenyi Biotec, 130090862) and MACS Separation LS columns (Miltenyi Biotec, 130042401) following the manufacturer’s instructions. 10 million isolated B cells were incubated with CellTrace™ Violet (CTV; Thermo Fisher Scientific, C34557) at 1:1,000 dilution at 37°C in the dark for 20 min, centrifuged at 300 × g for 10 min, and the cell pellet was washed once with 10 mL of MACS buffer. CTV-stained B cells were cultured in the B cell culture media (RPMI 1640 supplemented with 5% (v/v) FBS, 50 µM β-mercaptoethanol, 100 U/mL Penicillin G and 100 µg/mL streptomycin sulfate) at a seeding density of 1 million cells/mL. Cells were stimulated with 15 µg/mL lipopolysaccharide (LPS; Jomar Life Research, tlrl-3pelps) or with 1/1,000 recombinant CD40L plus 1/100 conditioned mouse IL4 supernatant for 96 h, and applied to flow cytometry analysis (BD LSRFortessa). Division and proliferation indices were determined using the cell proliferation module of FlowJo 10.3.

Teflon-glass dounce homogenizer
1.5 mL micro centrifuge tube
2 mL micro centrifuge tube
15 mL conical vial
Eppendorf 5424 centrifuge
Mitochondria Isolation Kit for mouse tissue, which includes anti-Tom22 microbeads, MACS separation LS Columns, and magnetic Quadro MACS Separator (Miltenyi Biotec, cat. no. 130-097-040).
Cell disruption vessel (Parr Instrument Company, cat. no. 4635)
Nitrogen tank
The Belly Dancer®, Belly Dancer shaker (Denville Scientific)

B cells were isolated from single-cell splenocyte suspensions using B-cell isolation kits (Miltenyi Biotec, 130090862) and magnetically activated cell sorting (MACS) separation LS columns (Miltenyi Biotec, 130042401) following the manufacturer’s instructions. Ten million isolated B cells were incubated with CellTrace Violet (CTV; Thermo Fisher Scientific, C34557) at 1:1,000 dilution at 37°C in the dark for 20 min and centrifuged at 300 × g for 10 min, and the cell pellet was washed once with 10 ml of MACS buffer. CTV-stained B cells were cultured in the B-cell culture media (RPMI 1640 [Sigma-Aldrich, R8758] supplemented with 5% (vol/vol) fetal bovine serum (FBS; Assay Matrix, ASFBS-F, batch A18C016], 50 μM β-mercaptoethanol, 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate) at a seeding density of 1 million cells/ml. The cells were stimulated with lipopolysaccharide (LPS; Invivogen, tlrl-3pelps), recombinant CD40L plus 1/100 conditioned mouse IL-4 supernatant (kind gift from Andreas Strasser, Walter and Eliza Hall Institute, Melbourne, Australia), or anti-IgM F(ab)₂ fragment (eBioscience 16-5092-85) at the concentrations and treatment times indicated in the figure legends and panels.