Countbright counting beads

We previously reported on the generation of Transmitted/Founder (TF) HIV-1 infectious molecular clones (IMCs) CH040.c and CH058.c [4 (link)]. TF IMC CH470 was generously provided by Beatrice Hahn (U. Pennsylvania) [69 (link)]. pCH040.c-GFP.T2A/K3223 (referred to herein as CH040.c-eGFP) was constructed from pCH040.c (GenBank #JN944939.1) by inserting eGFP using the nef ATG, followed by a self-cleaving T2A peptide cassette and nef in frame, analogous to a previous approach [70 (link)]. pCH470, pCH040.c and pCH058.c plasmids were prepared using Stbl3 cells (Invitrogen, Carlsbad, CA) and used to produce viral stocks in 293T cells as previously described [71 (link)]. Virus stocks were titered using an HIV-1 Gag p24 ELISA kit (Perkin Elmer, Waltham, Massachutsetts). Prevotella stercorea (DSM #18206, DSMZ, Braunschweig, Germany) stocks were prepared as previously detailed [35 (link)], frozen at -80°C in DPBS in single use aliquots and enumerated using CountBright counting beads and a LSRII flow cytometer (BD Bioscience, San Jose, CA).

Amounts of EMPs were measured using the flow cytometry method [4 (link),18 (link)]. In the assay, EA.hy926 cells were pre-treated with EA and Eth extracts (50–200 µg/ml) for 1 h and incubated with TNF-α (10 ng/ml) for 24 h. After incubation, the culture medium was collected and centrifuged at 5000×g for 15 min. The supernatant was collected and centrifuged at 20000×g for 40 min. The supernatant was discarded, and the EMPs pellet was reconstituted in Annexin V binding buffer and stained with FITC-Annexin V for 15 min in the dark at room temperature. Subsequently, CountBright™ counting beads were added in each sample as an internal control and 5000 counting bead events were analysed using FACSCalibur Flow Cytometer (BD) with BD CellQuest Pro Software (BD Biosciences, San Jose, CA, U.S.A.). For EMPs gating, calibrated latex beads (1.1 µm diameter) were used to set the population of particle size < 1 µm. The gated populations which stained with FITC-Annexin V were used to set the subpopulation of positive Annexin V EMPs. The numbers of positive Annexin V EMPs were normalised with the counting beads and were presented as absolute numbers of EMPs as well as the fold-change of EMPs relative to the control.