Dynabeads mouse t activator cd3 cd28 beads

Naïve CD4⁺ T cells were enriched from C57BL/6CNrl mice (Stemcell). Then, the cells were pre-treated with 0.5, 2.0, or 5 mM sodium acetate or vehicle (R10 medium) containing R10 medium for 4 hours. Dynabeads™ Mouse T-Activator CD3/CD28 beads were added according to manufacturer’s instructions for 4 hours (Thermo Fisher). At the conclusion of 4 hours, cells were centrifuged at 4°C at 200xg for 10 minutes and fixed with IC fixation buffer (Thermo Fisher) in 200 μL (100 μL IC fixation buffer + 100 μL PBS) for 15 minutes at 4°C. Following fixation, cells were washed with PBS three times and permeabilized with 1x eBiosciences™ permeabilization buffer for 1 hour at room temperature (Thermo Fisher). Phalloidin-iFluor 488 (Abcam) stock solution was diluted in PBS containing 1% w/v BSA 1000x. After permeabilization, cells were washed three times and Phalloidin-iFluor 488 solution added for 2 hours at RT.

CD8+ T cells were isolated from FVB/N mouse spleen using a CD8+ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). Spleen was minced and pressed through a strainer and washed with PBS. Collected cells were centrifuged for 10 min at 300× g. The pellet was resuspended in ACK lysis buffer (2 mL) for 10 min at 4 °C. Lysis was terminated with 30 mL PBS, and the suspension was then centrifuged for 10 min at 300× g. The pellet was resuspended with culture media (RPMI + 10% FBS). CD8+ T cells were isolated using magnetic cell sorting by negative selection using a CD8+ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). CD8+ T cells were activated using Dynabeads Mouse T-Activator CD3/CD28 beads (Thermo Fisher) using RPMI + 10% FBS for 24 h. Recombinant mouse IL-2 (30 U/mL) was added, according to the manufacturer’s protocol for T cell expansion. Activation status was confirmed by measuring IFN-ϒ and TNF-α release by ELISA.

Human CD4+ T cells were loaded with CTV before being stimulated with plate bound anti-CD3 (OKT3, 5 µg/ml) and soluble anti-CD28 (CD28.2, 1 µg/ml) in complete medium containing 30 U/ml IL-2 for 4 days. Cells were then stained with anti-TSAd-DyLight 488 and analysed by flow cytometry. Dividing cells were identified by CTV dilution. Murine CD4+ T cells were stimulated with Dynabeads® Mouse T-Activator CD3/CD28 beads (ThermoFisher), bead: cell ratio = 1:1 in complete medium containing 30 U/ml IL-2. CD3/CD28 beads were removed after 3 days in vitro and cultured in the presence of IL2 (30 U/ml) for another 7 days. Live cells were counted by trypan blue dye exclusion using a TC20 automated cell counter (Bio-Rad), and phenotyped by flow cytometry at 0, 3, 7 and 9 days in vitro. Surface staining was performed using CD4-PerCP-Cy5.5, CD44-FITC and CD62L-PE in FACS buffer (2% FCS, 0,1% sodium azide in PBS) at 4 °C. Cells were stained subsequently with LIVE/DEAD Fixable Near-IR to exclude dead cell, fixed for 10 minutes with 2% PFA in FACS buffer, and acquired on a FACSCantoII. Data was analysed using FlowJo version 7.6 (Tree Star Inc.), where live cells were gated on CD4-PerCP-Cy5.5 followed by analysis of CD44-FITC and CD62L-PE.

Total mouse T cells were labeled with CellTrace Violet (Invitrogen) for proliferation tracking. Following labeling, T cells (2 × 10⁵) were activated using Dynabeads mouse T-activator CD3/CD28 beads (Invitrogen) and co-cultured with 20% of conditioned media collected from typhoid toxin-treated macrophages (20% v/v) for 6 days. After the incubation period, T cell proliferation was assessed by performing surface staining with anti-CD4 and CD8 antibodies, and subsequently analyzed using flow cytometry.

CD3⁺ spleen T cells were isolated with Pan T cell isolation kit (Miltenyi, 130‐095‐130). CD4⁺CD25⁻ Teff and CD4⁺CD25⁺ Tregs were sorted by flow cytometry. Freshly sorted Teffs were stained with 5 × 10⁻⁶m CFSE (BD Bioscience) for 5 min at 37 °C. 5 × 10⁴ Teff cells were cocultured with Tregs at Teff/Treg ratios of 1:1, 2:1, and 4:1 in the presence of Dynabeads Mouse T‐Activator CD3/CD28 beads (Invitrogen, 11452D) and 30 U mL⁻¹ rhIL‐2 in RPMI complete medium (Gibco, Invitrogen). CFSE dilution was analyzed by flow cytometry after 72 h of culture. Detailed antibody information was listed in Table S3 (Supporting Information).

T cells were isolated from LNs by meshing through nylon filters and labeled with Cell Proliferation Dye eFlour 450 (eBioscience) according to manufacturer’s instructions. For the stimulation, T cells were incubated in full RPMI medium supplemented with IL-2 and Dynabeads Mouse T-Activator CD3/CD28 beads (Invitrogen) according to manufacturer’s instructions.