Cd45 conjugated microbeads

SupT1 cells stably transduced with individual dox-inducible IFITM members were infected with HIV-1 (2x10⁶ cells, MOI 0.5; 1 week in culture) or with VSV (2x10⁶ cells, MOI 0.05; 2 days in culture). At the end of the culture period, virions were harvested, syringe-filtered and divided in two fractions that were either left untreated or incubated with 50 μL of CD45-conjugated microbeads (Miltenyi, catalogue# 130-045-801) for 1 hour at 4°C in constant nutation. Microbeads were then passed through a retaining column (Miltenyi, catalogue#130-042-201) and the flow through was harvested. Virion particles contained in both CD45-depleted and non-depleted fractions were then purified by ultracentrifugation through sucrose, normalized by exo-RT activity and compared by WB and infectivity analyses. The anti-CD45 antibody used in WB was purchased by Becton Dickinson (catalogue# 610266).

Bone marrow samples were stratified on Ficoll Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA, catalogue number 10771) and centrifuged at 300 g for 30 min at 25°C. Mononuclear cells sedimented at the interphase were then collected, washed twice with PBS and assessed for viability by trypan blue staining (ThermoFisher, catalogue number 15250061). An average of 1 × 10⁸ bone marrow mononuclear cells (BM-MNCs) was labelled with CD34-conjugated microbeads (Miltenyi, Woking, UK) and immunomagnetically sorted. CD34-depleted cells were labelled with CD45-conjugated microbeads (Miltenyi) and further sorted. The CD34–CD45 double-negative population was labelled with CD146-conjugated microbeads (Miltenyi) and enriched through immunomagnetic sorting. The purity of the selected cell population was assessed by flow cytometry (see below). Samples with a purity below 90% were excluded from the study.
The CD34⁻CD45⁻CD146⁺ cell fraction was then seeded onto 24-well plates at a density of 1 × 10³ to 5 × 10³ cells per cm² and expanded in an α-MEM basal media (ThermoFisher Scientific, catalogue number 32561-029) supplemented with 20% FBS (ThermoFisher Scientific, catalogue number 16000044). Four to six cell lines per group were studied between passage three and seven in the subsequent experiments.

Human BM pericytes (BM-PCs) and stromal cells (BM-SCs) were obtained as previously reported [24 (link), 25 (link)]. Once approximately 90% confluent, BM-MSCs were trypsinised and sorted for isolation of PCs. Around 1 × 10⁷ BM cells were resuspended in pre-cooled column buffer containing 0.5% (w/v) BSA and 2 mM EDTA in DPBS, labelled with CD45-conjugated microbeads (Miltenyi) for 15 min at 4℃ before subsequently being sorted by magnetic separation. The CD45-negative cells were then labelled with CD34-conjugated microbeads (Miltenyi), followed by magnet sorting. CD45^neg CD34^neg cells were labelled with CD146-conjugated microbeads (Miltenyi). The CD34^neg CD45^neg CD146^pos population, collected through immunomagnetic sorting, was considered BM-PCs. The remaining cells (CD45^negCD34^pos and CD45^negCD34^negCD146^neg populations) were pooled and considered BM-SCs. The purity of BM-PCs was assessed by immunocytochemistry staining (data not shown). Cell lines between passage 3 to passage 6 were used for subsequent experiments.