Celltracetm violet

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).

The spleen was collected and immune cells were isolated according to an established protocol [43 (link)]. Immune cells were labeled with the fluorescent probe CellTraceTM Violet (CTV) (5 μmol/L, code C34557, Thermo Fisher Scientific) following the manufacturer’s instructions. CTV- Tcell were cultured in the absence or presence of Concanavalin A (ConA) (10 µg/ml) and total circulating sEVs (100 µg/ml) isolated from plasma obtained from women with normal glucose tolerance test (normal pregnancy) or gestational diabetes mellitus (GDM) in complete RPMI medium. After 4 days of culture, cells were stained with CD3-FITC (code 349201 Becton Dickinson), CD25-PE (code 341009 Becton Dickinson) and LIVE/DEAD™ Fixable Near-IR (code L10119, Thermo Fisher Scientific) and acquired on the flow cytometer Navios EX (Beckmann Coulter). The data were analyzed using the Beckman Coulter Kaluza Analysis Software. Percentage of proliferative viable CD3+ T cell was assessed by cell division based on CellTrace Violet fluorescence dilution following manufacturer’s instructions.

FACS-sorted CD44^loCD5^loLy6C⁻, CD44^loCD5^hiLy6C⁻, and CD44^loCD5^hiLy6C⁺ CD8⁺ T_N cells were labeled with 2.5 μM of Cell Trace^TM Violet (CTV; ThermoFisher). Each CTV-labeled subset was adoptively transferred into an individual sex and age matched Rag1^−/− recipient mice. Cells were isolated from mLN 7 days after transfer and analyzed by flow cytometry. For BrdU incorporation assay, mice were injected intraperitoneally (i.p.) with BrdU (1 mg/mouse) and then administered with drinking water containing BrdU (0.8 mg/ml) for 2−3 days before sacrifice. BrdU staining was performed with eBioscienceTM BrdU Staining Kit for Flow Cytometry FITC (eBioscience) according to manufacturer’s protocol. In brief, cells isolated from LI were stained with surface markers, then fixed and treated with DNase I. Cells were stained with fluorochrome-conjugated anti-BrdU antibody and analyzed with flow cytometry.

SIRPα⁺ (F4/80⁻CD64⁻CD11c⁺I-Ab⁺XCR1⁻) and SIRPα⁻ (F4/80⁻CD64⁻CD11c⁺I-Ab⁺XCR1⁺) DCs were sorted from B6 mouse spleens by flow cytometry, and pulsed with 1 nM or 100 pM OVA peptides (aa257-264). CD3⁺CD8⁺ T cells were sorted from OT-I mouse spleens by flow cytometry and labeled with Celltrace^Tm violet (Thermo Fisher Scientific). The OVA-pulsed DCs (1 × 10⁴) were co-cultured with Celltrace^TM violet-labeled OT-I CD8⁺ T cells (1 × 10⁵) for 3 days, and the cultured cells were analyzed for proliferation and activation by measuring violet fluorescence dilution and CD25 expression, respectively, using flow cytometry.

CellTrace^TM Violet was used for tracking proliferation in glioblastoma cell cultures, by fluorescent dye dilution and flow cytometry, according to the manufacturer's protocol (Molecular Probes, ThermoFisher, Fr).

After purification, T cells were labeled with 5 μM of either CFSE (Invitrogen) or CellTrace^TM Violet (Molecular Probes), as previously described (26 (link)) and injected i.v. into hosts. For inducing lymphopenia, normal B6 mice were treated with anti-Thy1.2 mAb 30-H12 (anti-Thy1.2) (Bio X Cell, i.p. injection in a single dose of 200 μg/mouse, 2 days before cell transfer) or 600cGy of whole-body irradiation (1 day before cell transfer). For generating antigen-induced “SP-like” response, SMARTA CD4⁺ T cells were transferred into the hosts, as indicated in the figures, followed by either LCMV Armstrong (2 × 10⁵ PFU) or LCMV peptide GP₆₁₋₈₀ (20 μg/mouse) through i.p. injection 1 day post cell transfer. OT-I CD8⁺ T cells were transferred into the hosts, as indicated in the figures, followed by immunization of OVA protein (Sigma Aldrich, 100 μg/mouse). HY.CD8⁺ cells from female HY mice were transferred into female hosts.