Ficoll paque

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque (STEMCELL Technologies Inc., Vancouver, Canada) density gradient separation from >10 mL peripheral fresh whole blood treated with EDTA K2 anticoagulant. The percentage of the CD4⁺CD25^high Treg population in CD4⁺ T cells was determined using flow cytometry analysis as described previously.^{[18 (link)]} In addition, intracellular staining of FoxP3 was conducted using fluorescently labeled antibody anti-CD3, anti-CD4, and anti-CD25 for surface maker staining, followed by fluorescein isothiocyanate (FITC)-anti-FoxP3 (eBiosciences, San Diego, CA) staining after permeabilization. Other fluorochrome-conjugated Abs-specific surface markers, including peridinin-chlorophyll-protein-anti-human leukocyte antigen - antigen D related, FITC-anti-CD45RA, and allophycocyanin-anit-CD45RO, were used to confirm the CD4⁺CD25⁺ Tregs as described in our previous study.^{[13 (link)]}

Peripheral blood samples were used to isolate the PBMCs by the density gradient centrifugation on Ficoll-Paque according to manufacturer's recommendation (StemCell Tech). Naive B cells were isolated by MACS negative selection with depletion of the CD2, CD14, CD16, CD27, CD36, CD43, CD235a positive cells according to manufacturer's protocol (Miltenyi). B-cell populations were sorted to the highest purity by using MACS positive selection of CD19 cells. Purity of sorted cells was monitored by ration of CD19⁺CD27⁻ to CD45⁺ by flow cytometry.

A549, H292, and H460 cells (5 × 10⁴ cells) were seeded in 24-well plates until 70–80% confluence under a cell culture condition. To isolate human PBMCs from peripheral blood donated by healthy volunteers, FicollPaque density centrifugation as well as Lymphoprep™ and SepMate™−50 (Stemcell Technologies, Vancouver, Canada) were used. The acquired PBMCs were seeded to a co-culture system at a PBMCs/attached NSCLC cell ratio of 5:1. The wells were treated with 5 µg/mL of anti-PD-1 mAb (nivolumab, #A1307; BioVision, Milpitas, CA, USA) or nobiletin (200 µM) or their combinations for 48 h. Then, the spend media were collected to analyze the human IFN-γ level, while the NSCLC cells viability was determined by MTT assay. The human IFN-γ were evaluated using an ELISA kit (#430104; BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol.

Human whole blood was collected (CIPO study, 2017–1506 approved by the ethics board of CIUSSS de l’Estrie CHUS) and processed immediately for PBMC using Ficoll-Paque (Stemcell). PBMC were resuspended at a concentration of 5 × 10⁶ in freezing media (RPMI with 12.5% Human Serum Albumin and 10% DMSO). 1 mL aliquots were frozen in cryovials overnight at − 80 °C and transferred to liquid N₂ for long-term storage. Following batch thawing of viably frozen cells, PBMC were stained with anti-human CD3, CD56, granzyme B, perforin and BODIPY 493/503 flow cytometry analysis. For cytotoxicity assessment, PBMC were rested for 20 h with recombinant IL2 (250 U). ⁵¹Cr-labelled K562 cells were then added and assessment of target cell killing was determined as above.

Peripheral blood (10 mL) was obtained for the isolation of monocytes. Monocytes were purified by density gradient centrifugation over Ficoll-Paque (Lymphoprep #07801, STEMCELL Technologies, Vancouver, Canada). Monocytes were incubated with PerCP conjugated mouse anti-human CD14 antibody (clone 61D3, eBioscience, Santiago, CA, USA). The proportion of TLR4 in this separated cell fraction was examined by FACS analysis using Mouse IgG2a IsoControl (eBioscience #53-4724-80) and Anti-human CD284 (TLR4) Clone HTA125 (eBioscience #53-9917-41) or Anti-human CD282 (TLR2) Clone T2.5 (eBioscience #25-9024-80). Cells were analyzed in a FACSCalibur (Becton-Dickson, NJ, USA) and were initially gated on the basis of forward and side scatter characteristics. Cell Quest Pro Software was used to analyze results. Results are expressed as relative and mean fluorescence intensity (rMFI).

PBMC were obtained from buffy coats of blood of healthy donors using a Ficoll-Paque density gradient centrifugation and monocytes were purified using negative selection antibody cocktails (StemCell Technologies) as described before [22 (link)]. Monocytes were cultured in complete culture medium (RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) or human serum (HS; Sigma) and penicillin/streptomycin (Gibco) and differentiated to monocyte derived macrophages (MDM) for 4 days in the presence of monocyte-colony stimulating factor (M-CSF, Peprotech) or granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech) both at 100 ng/ml. The protocol was approved by the scientific committee of Fundació IrsiCaixa. Buffy coats were purchased from the Catalan Banc de Sang i Teixits (http://www.bancsang.net/en/index.html). The buffy coats received were totally anonymous and untraceable and the only information given was whether or not they have been tested for disease. When appropriate, differentiated macrophages were incubated with 100 ng/ml of lipopolisaccaride (LPS, Sigma-Aldrich) overnight at 37°C.
TZM cells were received from the National Institutes of Health, AIDS Research and Reference Reagent Program. HEK293T cells were purchased from Dharmacon (Madrid, Spain).

Whole blood samples were collected from COVID-19 patients (total n = 24, mild n = 11, moderate n = 7, or severe n = 6 according to the WHO seven-point ordinal scale) admitted to the Royal Brisbane and Women’s Hospital (RBWH) processed within 4 h of collection. Patients were aged from 29 to 68 years of age. Following venesection, PBMCs were isolated from whole blood using Ficoll-Paque density gradient centrifugation in SepMATE PBMC isolation tubes (STEMCELL Technologies, Vancouver, Canada). Samples were then cytospun onto coverslips at a density of ~100,000 cells per coverslip and fixed with 3.7% formaldehyde. This study was performed in accordance with the NHMRC National Statement on Ethical Conduct in Human Research 2007 (updated 2018). Ethical approval was granted by the Royal Brisbane and Women’s Hospital Ethics Committee (HREC/2020/QRBW/63138) and accepted by the QIMR Berghofer (P3562). All participants provided written informed consent. Samples were stained for ACE2^me.