Apc cy7 cd8 ab

Spleen and draining lymph nodes were harvested from mice after rejection of graft or at experimental end point (> 200 days) if mice remained euglycemic up to that point. Organs were processed into single cell suspensions using frosted slides. ACK lysis buffer (ThermoFisher Scientific) was added to spleen to lyse red blood cells. Splenocytes were panned, labelled with (5(6)-Carboxyfluorescein N-hydroxysuccinimidyl ester) (CFSE), and used as responders in a standard mixed lymphocyte reaction assay [41 (link)]. Stimulator cells were prepared from either naïve BALB/c (donor) or C3H (3^rd party) mice, irradiated with 200 cGy, and cocultured with equal numbers of responder cells in 96-well plates (0.1×10⁶ cells/well). Cells were cultured in 200 μL DMEM supplemented with HEPES buffer, sodium pyruvate, penicillin/streptomycin, L-Glutamine (ThermoFisher Scientific), FBS, L-Arginine HCL, folic acid, L-Asparagine, 2-Mercaptoethanol (Sigma), and responder serum. Cells were harvested after four days of culture at 37°C and stained with Alexa 700-CD4 Ab, APC-Cy7- CD8 Ab, and 7AAD to separate dead cells (BD Pharmingen). Cells were collected using BD LSR II and analyzed using Diva software.

Spleen, kidney, and kidney draining lymph nodes were harvested from rejecting and long-term mice (> 200 days). Single cells were prepared from the spleen and lymph nodes by gentle mechanical dispersion and from islet-harboring kidneys by collagenase digestion. Cells were stained using antibodies to cell surface markers (Alexa 700-CD4 Ab 1:200 BD Bioscience 557956, APC-Cy7-CD8 Ab 1:100 BD Bioscience 557654, PE-Cy7-CD25 Ab 1:100 from Pharmingen 25-0251-82, BD, and eFlour 450-CD44 Ab 1:100 eBiosicences 48-0441-82 and PerCP-Cy5.5- CD62L Ab at 1:200 from eBioscience 45-0621-82). Intracellular FoxP3 staining was carried out on fixed/permeablized cells using FoxP3 Transcription Factor Staining Buffer set (diluted 1:20, eBioscience 11-5773-82). Data was collected using BD LSR II and analyzed using Diva software and FlowJo 9.8. Data was graphed using GraphPad Prism and t tests with Welch’s correction were used to determine significance between groups, p <0.05 was considered significant.