Celltracetm far red cell proliferation kit

Salmonella enterica serovar Typhimurium (STM) strain LT2 (ATCC 700220) and the clinical isolate STM-D23580, were used as representative non-typhoidal Salmonella sequence type 19 (ST19) and type 313 (ST313), respectively. Strain LT2 is one of the principal Salmonella laboratory strains used in cellular and molecular biology^{69 (link)}. Strain D23580 was isolated from the blood of an HIV-negative Malawian child with malaria and anaemia. Salmonella enterica serovar Typhi (ST) strain Ty2 (ATCC 700931) was used as representative typhoidal serovar. All bacteria were grown to logarithmic growth phase in LB Lennox broth (Sigma) supplemented with sucrose (Sigma) at a final concentration of 10%. Aliquots were kept frozen at –80°C until use, while bacterial viability was monitored periodically. For each experiment, an aliquot of bacteria was thawed, diluted in RPMI 1640 (Sigma) and incubated in the presence of 5uM of CellTrace^TM Violet or CellTrace^TM Far Red Cell Proliferation kit (ThermoFisher) at 37°C for 20 min while shaking (200 rpm). Bacteria were then washed and re-suspended in RPMI to obtain a multiplicity of infection (MOI) of 10:1. The number of microorganisms was assessed at each time point post infection by plating 10-fold dilutions of the bacterial suspension, in quadruplicate, on LB Lennox agar (Sigma). The number of bacteria was determined as colony forming units (CFU).

ZsGreen tagged BTSC#233 cell lines were cultured as previously described by us^{37 ,72 (link)}. Post trypsinization, a centrifugation step was performed, following which the cells were harvested and suspended in MEM media at a concentration of 20,000 cells/µl. inoculate Cultured tissue sections were inoculated with 1 µl of tumor cells using a 10 µl Hamilton syringe, and further cultured as required. Matched peripheral blood samples were collected from the cortical tissue donors. Peripheral T-cells were isolated using the same MACSxpress^® Whole Blood Pan T-Cell Isolation Kit (Miltenyi Biotech, Bergisch-Gladbach, Germany) and erythrocytes were eliminated from the suspension using ACK-lysis buffer (Thermo Fisher Scientific, Carlsbad, USA). T-cells were then fluorescently tagged using the CellTrace^TM Far Red Cell Proliferation kit (C34564, Thermo Fisher Scientific, Carlsbad, USA) prior to inoculation. Endogenous IL-10 receptor expressed on T-cells were blocked using anti-IL10 neutralizing antibody (ab34843, Abcam, Cambridge, UK) at a concentration of 5 µg/ml for 1 h at 37 °C. Both naïve and neutralized T-cells (40,000 cells/µl) were inoculated into tissue sections and cultured for 48 h.