Celltrace cfse cell proliferation kit for flow cytometry

To assess the effect of liposomes loaded with everolimus on LFs derived from CLAD and CTD-ILD patients, we used CellTrace™ CFSE Cell Proliferation Kit for flow cytometry (Thermo Fisher Scientific, Monza, Italy). LFs were seeded on 12-well plate at a density of 3 × 10⁵ cells after labeling them with CFSE dye following the manufacturing instruction. After 24 h, cells were incubated with PEG-LIP(ev), PEG-LIP(ev)-HA400kDa and everolimus alone. In this case, liposomes were added with the same concentration of everolimus (50 nM). After 24, 48 and 72 h cells were harvested and analyzed by flow cytometer to quantify the fluorescent signal of CFSE dye.

Cells were incubated at 37°C, 5% CO₂ in RPMI 1640 (Gibco, Thermofisher, Waltham, MA, USA) supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco) in 96-well plates. For the proliferation assay, total PBMCs or purified T cells were pre-stained with Carboxyfluorescein 6 succinimidyl ester (CFSE) prior to the culture. CellTrace™ CFSE Cell Proliferation Kit, for flow cytometry (C34554, Thermofisher) was used for CFSE staining. Five million cells (or sometimes a lower number) were stained in 1 ml CFSE staining buffer (2 μM, FCS-free) for 10 min at 37°C. After washing, 0.2 million of CFSE-labeled PBMCs, or 0.1 million of CFSE-labeled T cells, were seeded into a flat 96-well plate. Dynabeads^® Human T-Activator CD3/CD28 (11161D, Thermofisher), soluble anti-CD100 (see above), soluble anti-CD72 (3F3, Biolegend, San Diego, CA, USA), CD72-Fc proteins (Recombinant Human CD72 Fc Chimera Protein, R&D, Minneapolis, MN, USA), or matched isotypes were added as indicated. The dilution of CFSE fluorescence was recorded at day 3 and/or day 5 in the FITC channel on the flow cytometer. The bead-to-cell ratio was 1:2 in the whole PBMC culture or 1:1 in purified T cell culture.

Cells were extracted by dissolving the microspheres in 55 mM Sodium Citrate and 10 mM EDTA in PBS for 10 min at 37°C. Cells were then suspended in PBS. Surface and intracellular staining was done in a three-colour analysis with combinations of fluorescein isothiocyanate (FITC), phycoerythrin (PE) and allophycocyanin (APC). Antibodies used included anti-CD3, anti-CD4, anti-CD8, anti-CD14 and anti-CD68 (ImmunoTools, Germany). For T–cell proliferation, PBMCs were stained with CellTrace CFSE Cell Proliferation Kit for flow cytometry (ThermoFisher Scientific, UK) before infection with Mtb. Fluorescence was then analyzed by flow cytometry (BD Accuri C6 flow cytometer).

HepG2 target cells were labelled using the CellTrace™ CFSE Cell Proliferation Kit for flow cytometry, following the manufacturer’s instructions (Molecular Probes). CFSE-labelled HepG2 target cells were plated at 40:1, 20:1,10:1, 5:1, 2.5:1, and 1.25:1 or at a 1:0 ratio with effector NK cells for 4 h, and target cell viability was detected by fluorescence with the 7AAD dye (Miltenyi Biotec, dilution 1:10). NK cells were counterstained with APC-CD56 (IgG1, clone TULY56, from eBioscience, San Diego, CA, USA, dilution 1:10).