Fix and perm

To assess the ImageStream analysis, which combines high-resolution microscopy and flow cytometry, 4 × 10⁶ THP-1 cells, previously treated with SerpinB3 (100 ng/mL) in the presence of RAP (5 g/mL) or a medium alone for 24 h, were fixed and permeabilized with Fix and Perm (Invitrogen Life Technologies, Waltham, MA, USA), blocked with 5% goat serum (Invitrogen Life Technologies, Waltham, MA, USA) in PBS containing 1% BSA, and incubated with anti-LRP-1 for 1 h at room temperature. After washing, the cells were incubated for 30 min with secondary antibodies anti-mouse Alexa Fluor 488. Cellular nuclei were counterstained with Dapi by 3 min incubation (Merck KGaA, Darmstadt, Germany). For ImageStream analysis, 60 L of each sample was used, and the results were assessed by the IDEAS software (Luminex, Genk, Belgium).

PHA-blast target cells from 10 HIV-uninfected donors, either BAGN-treated or -untreated, and stained for surface activation markers and HIV co-receptors expression, were further used for the VRAs. At day 3, both BAGN-treated and -untreated target cells were infected (MOI = 0.001) with the viral stocks produced with or without BAGN in the culture medium. Cells were cultured for 7 days in R20/50, with or without 0.2 mM BAGN, at 37°C in a 5% CO₂ containing atmosphere. Secretion of p24 into the culture supernatant was measured by ELISA (Fujirebio) and intracellular p24 production was measured by flow cytometry. Briefly, cells were stained with Live/Dead violet fluorescence fixable dead cell stain reactive kit (Invitrogen) followed by extracellular staining with antibodies: CD3-APC-H7, CD4-PreCp, and CD8-APC (BD Biosciences). The cells were fixed and permeabilized (Fix and Perm, Invitrogen) while staining for HIV Gag p24 using the KC57-FITC antibody (Coulter). As before, cells were collected on an LSR II instrument (Becton Dickinson) and analysis performed using the FlowJo software.

Benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside-treated and -untreated PHA-blasts were used to monitor expression of surface markers of cell activation and HIV co-receptor CCR5 and CXCR4. Briefly, PHA-blasts were prepared from 20 HIV-negative donors by stimulation with PHA for 2 days. One-half of the cells from each sample was then treated with 2 mM of BAGN overnight. The next day, cells were stained for viability markers (Live/Dead Fixable Dead Cell Stain kit, Invitrogen) and markers of T cell activation (antibodies: anti-human CD3-APC-H7, CD4-PE-Cy7, CD8-V500, CD25-APC, HLA-DR-FITC, and CD38-PerCP-Cy5.5; BD Biosciences). Cells were fixed and permeabilized (Fix and Perm, Invitrogen) to stain with Ki67-PE (BD Biosciences). Additional aliquots of BAGN-treated and -untreated PHA-blasts were used to stain for viability and T cell subsets as before and for HIV co-receptors (antibodies: anti-human CD184-APC (CXCR4), CD195-PE (CCR5); BD Biosciences). Cells were collected on an LSR II instrument (Becton Dickinson) and analysis was performed using FlowJo software.

Recombinant EBOV GP ΔTM (IBT Bioservices) was coated onto a MaxiSorp 96 well plates (Nunc) at 300 ng/well at 4°C for 18 hr. Wells were washed three times with PBS and blocked with 5% bovine serum albumin in PBS. Antibodies were diluted to 5 μg/mL in PBS, and added to the plates, and were incubated for an additional 2 hr at 37°C. Unbound antibodies were removed by washing three times with PBS, and human NK cells freshly isolated from peripheral blood of human donors by negative selection (Stem Cell Technologies, Canada) were added at 5 × 10⁴ cells/well in the presence of 4 μg/mL brefeldin A (Sigma Aldrich) and 5 μg/mL GolgiStop (Life Technologies) and anti-CD107a antibody (PE-Cy5, Clone H4A3, BD Biosciences). Plates were incubated for 5 hr at 37°C. Cells were stained for NK cell markers (CD56 PE-Cy7, clone B159, BD Biosciences; CD16 APC-Cy7, clone 3G8, BD Biosciences; CD3 Alexa Fluor700, clone UCHT1, BD Biosciences), followed by fixation and permeabilization with Fix and Perm (Life Technologies) according to the manufacturer’s instructions to stain for intracellular IFNγ (APC, Clone B27, BD Biosciences) and MIP-1β (PE, Clone D21-1351, BD Biosciences). Cells were analyzed on a BD LSRII flow cytometer. The glycan cap-specific mAb c13C6 (IBT Bioservices) was used as a positive control, and the HIV-specific mAb 2G12 (Polymun Scientifics, Austria) was used as a negative control.