Trypan blue exclusion

Cell viability was determined by trypan blue exclusion (Life Technologies) and caspase-cleaved CK18 immunostaining (cytoDEATH M30, 0.5 μg/ml, Roche) according to the manufacturers' protocols. Briefly, a 0.4% solution of trypan blue was added to the cell suspension at 0.04% final concentration. Blue-stained cells and total cells were counted immediately under a low-power microscope. Caspase-cleaved CK18 immunostaining was performed as previously described [30 (link)].

Spleens from naïve and P. chabaudi AS infected B6 mice were collected aseptically at the indicated times post-infection (p.i.). To prepare single cell suspensions, tissues were perfused with PBS containing 1% FCS (HyClone Laboratories, Logan, UT), teased apart, and gently pressed through a sterile fine wire mesh. The cells were centrifuged, re-suspended in 0.175 M NH₄Cl to lyse red blood cells (Sigma-Aldrich, St. Louis, MO), washed, and re-suspended in complete RPMI 1640 medium (Life Technologies, Burlington, ON, Canada) supplemented with 5% heat-inactivated FCS, 25 mM HEPES (Life Technologies), 0.12% gentamicin (Life Technologies), and 2 mM glutamine (Life Technologies). The total number of spleen cells obtained from individual mice was determined using a hemocytometer. Cell viability was determined by trypan blue exclusion (Life Technologies) and was always >95%. For some experiments, CD4⁺ T cells were purified from single cell suspensions prepared from spleens of naïve and infected mice at the indicated times p.i. using a negative CD4⁺ T cell isolation kit (Miltenyi Biotec) following the manufacturer's instructions.

Effects of compound treatment on cellular metabolism was assessed by an XTT-based in vitro toxicology assay kit (Sigma-Aldrich) or Trypan blue exclusion (Life Technologies) as proxy for degree of cytotoxicity and expressed relative to DMSO control treatment. For measurement of cell viability using XTT, culture media was removed after 24, 72, and 96 h of compound treatment, replaced with 20% XTT solution and incubated at 37 °C in a 5% CO₂ humidified incubator for 2–6 h, and relative cell viability was measured in compound treated cells relative to DMSO-treated cells. Cell viability measurements in CEM-GXR cells are described above.

Cytotoxicity of GPS491 was assessed by using a CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA), alamarBlue or Trypan blue exclusion (Life Technologies, Waltham, MA, USA) and expressed relative to cells treated with DMSO (1%) alone. alamarBlue was added to culture medium prior to harvest/fixation, cells were incubated at 37 °C in a 5% CO₂ humidified incubator for 2–6 h, and fluorescence reflecting cell metabolic rate was measured using Bio Tek Cytation 5 (Winooski, VT, USA or TECAN infinite 200Pro fluorescence plate reader (Meilen, Zurich, Switzerland) (fluorescence detection, 560 nm (ex)/590 nm (em)).

HIV-1-infected T cells were preincubated with 100 μM 2-aminoethyl diphenylborate (2-APB) (Merck), 5 μM oligomycin (Sigma), 1 μM nocodazole (Sigma), 50 μM Mdivi (Sigma), 10 mM EGTA (Fluka), 10 μM BAPTA-AM (Life Technologies), or 0.5% dimethyl sulfoxide (DMSO) as a control for 30 min prior to mixing with target T cells. 2-APB was removed by washing after treatment of HIV-1-infected cells. Infected cells were then mixed with target cells and processed for immunofluorescence microscopy as described above. Cell-free virus production was quantified with an enzyme-linked immunosorbent assay (ELISA) to measure p24 (35 (link)). Viability and ATP concentration of the treated cells were measured by trypan blue exclusion (Life Technologies) and CellTiter-Glo (Promega), respectively.