Cell Reports, Volume 23

Supplemental Information

Progressive Upregulation of Oxidative Metabolism

Facilitates Plasmablast Differentiation
to a T-Independent Antigen
Madeline J. Price, Dillon G. Patterson, Christopher D. Scharer, and Jeremy M. Boss
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Cell Isolation
Splenic cell suspensions were made by mechanically forcing spleens through a 40 µm filter and lysing red blood
cells with ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 3 mins before quenching the
reaction with four volumes RPMI 1640 media (Corning Cellgro 50-020-PC) supplemented with 10% heat-
inactivated FBS (Sigma-Aldrich), 1% MEM non-essential amino acids (Sigma ENBF3930-01), 100 µM sodium
pyruvate (Sigma RNBF6686), 10 mM HEPES (HyClone SH30237), 1x Penicillin-Streptomycin-Glutamine (Gibco
10378016), 0.0035% β-mercaptoethanol (Sigma-Aldrich). Naive B cells were isolated using immunomagnetic
negative selection for CD43 (Miltenyi #130-090-862) following the manufacturer's protocol. Purity was confirmed
by flow cytometry. Plasmablasts were isolated by staining with CD138-APC (BD, 281-2), and enriching the
CD138+ fraction of splenocytes using a positive immunomagnetic enrichment on APC (Miltenyi # 130-090-855).
Purity greater than 80% CD138+ was used in downstream analyses (Seahorse). GL7+ cells were subsequently
enriched from CD138 depleted sample in order to avoid contaminating plasmablasts that are CD138+ and also
express GL7. CD138 depleted cells were stained with GL7-PE (Biolegend #144608) then enriched using a positive
immunomagnetic enrichment on PE (Miltenyi #130-105-639).

Flow Cytometry
Staining panels included anti- Fc (anti-CD16/CD32) (Tonbo Biosciences, 2.4G2), anti-CD11b (Tonbo Biosciences
M1/70), anti-CD11c (Tonbo Biosciences N418), anti-F4/80 (Biolegend BM8), and Thy1/2 (Biolegend 30-H12)
conjugated to APC-Cy7 each at a concentration of 0.25 µg per 1 × 106 cells to remove dendritic cells, macrophages,
and T cells. The following stains and antibody-fluorophore combinations were used to assess cellular phenotype:
anti-B220-PerCP-Cy5.5 or -PE-Cy7 (Tonbo Biosciences RA3-6B2) at 0.5 µl per 1 × 106 cells; anti-CD138-APC, or
-BV711 (BD, 281-2) at 0.125 µl per 1 × 106 cells; anti-GL7-BV421 or -PE (Biolegend GL7) at 0.25 µl per 1 ×
106 cells; Zombie Yellow (BV570) (Biolegend 77168), and CTV (Life Technologies #C34557). CTV was used at 10
µM per 1 × 107 cells/ml. Intracellular IgM staining was performed using the BD Fixation/Permeabilization Kit
(554714) using IgM-FITC at 0.25 µl per 106 cells (eBioscience 1-5890-85). Reactive oxygen species and superoxide
were measured with ROS/Superoxide Detection Assay (Abcam 139476) as per manufacturer’s protocol. This kit
included the inhibitors N-acetyl-l-cysteine and pyocyanin. Staining panels included fluorescence minus one (FMO)
controls to ensure that correct compensation and gating were applied.

Ex vivo differentiation
B cells were differentiated ex vivo as previously described37, but with the incorporation of division-tracking dye,
Cell Trace Violet (CTV). B cells were isolated using immunomagnetic separation as above, and stained with CTV at
a concentration of 20 × 106 cells/ml in PBS. Cells were differentiated at an initial concentration of 0.5 × 106 cells per
ml with LPS (20 µg/ml; Sigma #L2630), IL-2 (20 ng/ml; eBioscience #14-8021), and IL-5 (5 ng/ml;
eBioscience #14-8051). Half doses of LPS and cytokines were given on subsequent days. Where indicated,
dichloroacetate (Sigma 347795) was dissolved in dH2O and added at 10 mM concentration each day of culture.
Seahorse Bioanalyzer
Cartridge of FluxPack was hydrated at least 12 hours prior to running assay with 200 µl dH 2O in 37°C, non CO2
incubator. 1 hour prior to assay, dH2O was removed and 200 µl pre-warmed Seahorse calibrant solution (Agilent
103059-000) was added to wells. Purified cell populations were washed in Seahorse XF Assay media supplemented
with 1 mM sodium pyruvate, 2 mM L-glutamine (Sigma G7513), and 5.5 mM glucose for Mitochondrial Stress
Test, before adjusting the pH to 7.4 +/- 0.1 at 37°C. For Glycolytic Flux Test, Seahorse XF Base Media without
phenol red (Agilent 103335) was supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 5 mM HEPES
(HyClone SH30237), and 5.5 mM glucose. Cells were washed 1x in appropriate media and counted by flow
cytometry with AccuCheck counting beads (Invitrogen PCB100). CellTak (Corning 354420) was diluted in sterile
PBS to a final concentration of 22.4 µg/ml. 25 µl was added per well of Seahorse XFe96 cell culture plate and
incubated for 20 mins at room temperature before use. CellTak was washed out with 200 µl dH2O and then 400,000
cells were plated per well. Cell plate was incubated in 37°C, non CO2 incubator for 45 minutes prior to running
assay. Drugs were diluted in Seahorse prepared media for injection into each port. Port A, Oligomycin (Sigma
75351), was used at a final concentration of 2 µM. Port B, FCCP (Sigma C2920) was used at a final concentration of
2.5 µM. Port C injects a combination of Rotenone (Sigma R8875) and Antimycin A (Sigma A8674), each at a final
concentration of 1 µM. 2-DG (D8375) was used at a final concentration of 50 mM for Glycolytic Rate Assay.

Adoptive Transfer
µMT mice were bred to CD45.1+ C57Bl/6 mice to generate CD45.1+ µMT mice. Splenocytes were isolated from
CD45.2+ Blimpfl/fl Cd19Cre/+ or Cd19+/+ mice, labeled with Cell Trace Violet (ThermoFisher C34557) as per
manufacturer’s instructions, and injected via tail vein into sex-matched CD45.1+ µMT host mice. 24 hours later,
host mice were infected with 50 µg LPS (Salmonella Minnesota R595 Enzo ALX-581-008) via tail vein injection.
Mice were analyzed three days following LPS challenge.