Facsflow

Peripheral blood mononuclear cells (PBMCs) from TB patients and healthy controls were isolated from whole blood using Ficoll density gradient centrifugation, as previously described [19 (link)]. Bronchoalveolar cells (BALc) from TB patients were processed in a BSL3 laboratory. Bronchoalveolar lavages were treated with Sputasol (Oxid) for 15 minutes at room temperature. After filtering with BD Falcon cell strainer 100 μm, BALc were washed twice in PBS 1X and suspended in FACSFlow (BD-Pharmagen). The following anti-human Abs were used: anti-CD11b (clone ICRF44), anti-CD14 (clone HCD14), anti-CD15 (clone W6D3), anti-CD33 (clone HIM3-4), anti-HLA-DR (clone L243), anti-CD3 (clone UCHT1). PBMCs and BALc were incubated for 15 min at 4°C with fluorochrome-conjugated mAb. Samples were washed in PBS 1X, fixed in paraformaldehyde (PFA 1% for PBMCs and 4% for BALc), suspended in FACSFlow and immediately acquired using FACS CANTO II flow cytometer (BD Biosciences). A total of 500,000 events were acquired for each sample and analyzed with CellQuest software (BD Biosciences). The HLA DR- CD11b+ CD33+ cells were sorted (FACS ARIA II, BD Biosciences) and cytospin slides of enriched cell fractions were prepared and stained with hematoxylin/eosin dye according to standard protocols.

Following above‐described coculture regimes, DCs were harvested by pipette aspiration. The chondrogenic hBMSC pellets were removed prior to the DC harvest. DCs were centrifuged for 8 min at 248g. The cells were resuspended in FACSflow (BD Biosciences), and 2 × 10⁵ cells per sample were transferred to FACS tubes. Cells centrifuged for 5 min at 689g were resuspended in 100 μl of FACSflow containing anti‐CD11c (clone B‐ly6; allophycocyanin), anti‐HLA‐DR (clone G46‐6; peridinin chlorophyll protein), anti‐CD86 (clone 2331; phycoerythrin), anti‐CD80 (clone L307.4; phycoerythrin‐Cy7), anti‐CD14 (fluorescein isothiocyanate [FITC]) antibodies (all BD Biosciences), and live and dead cell marker (Life Technologies; APC‐Cy7) and incubated for 30 min at 4 °C in the dark. Samples were washed twice with FACSflow (centrifuged for 5 min at 689g) and resuspended in 200 μl of FACSflow. Samples were analysed on a FACSJazz (BD Biosciences) and FlowJo software version 10.0.7 (Treestar Inc.).

HUVEC were incubated with TNFα (25ng/ml) and two ratios of MP (1:50,000, 1:100,000 HUVEC : MP) during 24h. Then, the cells were trypsinized and washed with FACS Flow (BD Biosciences, San Jose, CA). The immunophenotypic characterization of the activation state of endothelial cells was done by incubating HUVEC with mouse-anti-human monoclonal antibodies against CD54-APC, CD106-BV421, CD62e-PE, CD31-FITC, VEGFR2-PE, CD105-FITC and TIE2-Alex647 (all BD Biosciences). All the antibodies were incubated with the cells for 30 min, at room temperature in the absence of light. After two washes with FACS Flow, flow cytometric analysis was performed using FACSCANTO-II with FACSDIVA Software (BD Biosciences).

Surface staining (phenotyping) was performed using fresh peripheral blood collected in the lavender tube with EDTA, BD Vacutainer. A 150-μL sample of fresh blood was used for each test tube (Th1, Th17, Treg,). Blood was incubated with a specific antibody mix for each population, for 30 min in the dark.
Th1: FITC anti-human CCR5 (BD), PE anti-human CXCR3 (Biolegend), APC anti-human CD4 (BD);
Th17: FITC anti-human CCR6 (Biolegend), PE anti-human CD161 (BD), APC anti-human CD14 (BD);
Treg: FITC anti-human CD4 (BD Pharmigen), PE anti-human CD25 (BD Pharmigen), APC anti-human CD127 (Biolegend).
Subsequently, FACS lysing (BD) was added to remove red blood cells (15 min, in darkness at room temperature). Finally, cells were washed with cold FACS Flow (BD) then resuspended in 1 mL of FACS Flow (BD). Ten thousand gated CD4+ cells were acquired by Cell-Quest four colors Facs Calibur cytometer (BD). The analysis of the results was carried out using the FlowJo software v.8.4.

The phenotype of effector T cell populations was determined by FACS analysis using antibodies described above, as well as mouse- antisheep or antibovine MHC class I (MCA2444GA; AbD Serotec), B-cell (MCA2443GA; AbD Serotec) and CD8 (MCA2216GA; AbD Serotec). Cells (2 × 10⁵–1 × 10⁶) were mixed with an equal volume of primary antibody (final concentration 1 μg ml^-1), incubated at 4°C for 30 min, washed three times using PBS, and resuspended in 50 μl FACS medium (2% horse serum in PBS). FITC-labelled goat anti-mouse IgG (AbD Serotec) was used as secondary antibody. Cells were incubated at 4°C for 30 min, washed as before and resuspended in FACSFlow (Becton Dickinson Biosciences) for data acquisition and analysis using either a FACSCalibur (Becton Dickinson Biosciences) or a FACSAria (Becton Dickinson Biosciences).

HIV-infected Taiwanese patients receive HIV care according to the national treatment guidelines at designated hospitals around Taiwan by the Centers of Disease Control in Taiwan. Residual plasma samples for routine determination of plasma viral load (PVL) were obtained from HIV-1-infected patients seeking HIV care at the NTUH. A standardized case record form was used to collect information on demographics, antiretroviral medications, baseline CD4 lymphocyte count, and PVL. PVL and CD4 count were quantified with the use of Cobas Amplicor HIV-1 Monitor^TM Test, version 1.5, (Roche Diagnostics Corporation, Indianapolis, USA) and FACSFlow (Becton Dickinson), respectively. The study was approved by the Research Ethics Committee of the National Taiwan University Hospital, Taipei, Taiwan (REC registration number, 200905045R) and the experiment procedures were carried out in accordance with the approved guidelines. The patients who were linked to HIV care at the hospital gave written informed consent for determination of HIV-1 resistance mutations.