C36950

Three independent cultures of trypanosomes were treated with I-BET151 for 3 d or with DMSO as a control. Following treatment cells were washed, counted on a hemacytometer and then serially diluted to a concentration of 5 cells/100 μL HMI9 or 50 cells/10 0μL HMI9 with no drug. They were plated on three 96-well plates and cultured for 3–4 d at 37°C before quantifying the number of wells growing in each plate. An aliquot of each culture was subjected to PI staining (BD 51–66211) at 0.75 μg/ml to assess viability and countbright beads (Life technologies C36950) were added to quantify cell growth. Washed cells were allowed to recover for 3 d before being subjected to PI staining again.

Meningeal and spleen single-cell suspensions were pipetted into a 96-well plate and pelleted. Cells were treated with 50 μL of Fc block (0.1% rat gamma globulin [Jackson ImmunoResearch], 1 μg/mL of 2.4G2 [BioXCell]) for 10 min at room temperature. Cells were then stained for surface markers and incubated with a fixable live/dead viability dye for 30 min at 4 °C using the following surface markers at a concentration of 1:200: CD45.2 EF450 (30-F11, Thermo Scientific), B220 PE-Cy7 (103222, BioLegend), and CD19 FITC (11-0193-81, eBioscience). Fixable Viability Dye eFluor 506 (65-0866-18, eBioscience) live/dead dye was used at a 1:800 dilution. Splenocytes were stained with the live/dead viability dye and were used for compensation controls. Finally, samples were resuspended in FACS buffer and acquired on a Gallios flow cytometer (Beckman Coulter). Cell counts were determined using absolute counting beads (Life Technologies, C36950) pipetted into samples just prior to acquisition. Data analysis was performed with FlowJo software v.10.

To quantify T cells in ear skin, dermal sheets were separated, and finely minced in RPMI 1640 media (ThermoFisher 11875093) complemented with 10% fetal bovine serum (R&D Systems S11150) (cRPMI) containing 100 μg/mL of Liberase TL (Roche 5401020001) and 50 μg/mL of DNase I (Sigma-Aldrich DN25). Minced tissue was incubated with shaking at 37°C for 1 h and then strained through a 70 μm filter into a new tube containing 1 mL cRPMI. Cells were stained with cell surface stains and live-dead stain at 4°C for 15 min in PBS. Flow cytometry was then performed using an LSR II or LSR Fortessa instrument (BD Biosciences). Compensation was performed using compensation beads (BD Biosciences 552845). Flow cytometry data was analyzed using FlowJo software (BD Biosciences). Staining antibodies used included CD45.2 (mouse, PE fluorochrome, clone 104, BD Biosciences 560695, 1:200 dilution), TCRβ (mouse, PE-Cy7 fluorochrome, clone H57-597, BioLegend 109222, 1:200 dilution), CD4 (mouse, FITC fluorochrome, clone RM4-5, BioLegend 100510, 1:200 dilution), CD8a (mouse, PerCP-Cy5.5 fluorochrome, clone 53–6.7, BioLegend 100734, 1:200 dilution) and Live/Dead Near-IR (ThermoFisher L10119, 1:1000 dilution). CountBright beads were used for counting cells and normalization (ThermoFisher C36950).