Lsr 2 flow cytometer

PLGG tumor cells were labeled with APC-conjugated human CD133 antibody and FITC-conjugated human CD15 antibody (Miltenyi Biotec), or isotype control antibodies at 4°C for 15 minutes in FCM buffer comprised of DPBS, 0.5% BSA and 2 mM EDTA. After washing, cells were re-suspended in FCM buffer containing 2 μg/mL propidium iodide (PI) and analyzed with a LSR II flow cytometer and Kaluza Analysis Software Version 1.3 (Beckman Coulter). Dead cells were excluded by PI staining.

The following antibodies were used to stain PCW cells: B220 PerCP-Cy5.5 (BioLegend, 103236), CD5 PE-Cy7 (BioLegend, 100622), CD19 BUV395 (BD Biosciences, 563557), IgM APC (Biolegend, 406509), PD-L2 BV421 (BD Biosciences, 564245), CD11b APC-Cy7 (BioLegend, 101226), Live/dead Ghost violet 510 (Tonbo Biosciences, 13-0870). For cells isolated from PZTD^+/+ mice, ZsGreen fluorescent protein was analyzed using the same channel as FITC. For cells isolated from CD19-Cre^+/- PTZD^+/+ mice, TdTomato fluorescent protein was analyzed using the same setting for TexasRed. To analyze TILs, CD45 PerCP-Cy5.5 (eBioscience, 45-0451-80), was added to the above panel replacing B220 for leukocyte gating. The stained samples were analyzed at the Boston University Medical Campus Flow Cytometry core facility using a BD LSRII flow cytometer and a Beckman Coulter MoFlo for cell sorting. Live leukocytes were gated by forward and side scatters, live/dead Ghost violet 510 and CD45 staining. B1 cells were gated as B220IntCD5Int population from the live leukocyte gate. L2pB1 cells were gated as PD-L2+IgM+ from the B1 cell gate. In the transgenic-knock in mice, L2pB1 cells were also identified by the expression of ZsGreen and TdTomato (Supplementary Figure S1).

Male C57BL6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) at 8 weeks of age. Mice were housed in the institutional animal care facilities at the IU Medical School with a strict 12 h:12 h light/dark cycle. Diabetes was induced with an injection of streptozotocin (STZ) (50 mg/kg). Animals were confirmed to be diabetic after 1 month of housing and when the serum glucose level was above 13.9 mmol/l for at least two consecutive measurements. At 4 months of age, diabetic and control mice were randomly assigned to a time point of a 24 h cycle and terminated at ZT (‘zeitgeber’; hours since ‘lights on) times ZT1, ZT5, ZT9, ZT13, ZT17 and ZT21. Single-cell suspensions were prepared from whole blood at the termination of the experiment. Samples were fixed and data were acquired on an LSR II flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA). Further details are provided in the ESM Methods.