Cd86 pe cy7

Non-specific binding sites on harvested cells were blocked with 20% inactivated AB-serum in PBS containing 0.1% BSA and 0.1% NaN₃ for 20 min at 4 °C. Then, cells were stained for 30 min at 4 °C with fluorescence-labelled antibodies: CD83-FITC, CD19-PECy7, CD80-APC647, CD14-APCCy7, HLA-DR-Brilliant Violet 421 (all Biolegend, San Diego, CA) CD86-PE, CD86-PECy7 (both BD Biosciences) and fixable viability dye eFluor506 (eBioscience, San Diego, CA, USA). Fluorescence was assessed by flow cytometry on a BD FACS Canto II and analyzed with FlowJo software.

After in vitro generation of DCs, NCI-H1975, A549 and NCI-H1650 cells were labelled with PKH67, a green fluorescent membrane dye (Sigma Aldrich, MIDI67), prior to plating them out on day 3. NSCLC cells were treated with chemotherapy on day 4. On day 5, immature DCs were stained with cytoplasmic violet-fluorescent CellTracker Violet BMQC dye (Invitrogen, C10094) and effector (E) and target (T) cells were placed in coculture at a 1:1 (E:T) ratio. Supernatant (SN) was stored (−20 °C), cells were collected and immediately used for flowcytometric detection of DC maturation markers and phagocytosis on day 7. Cells were stained with CD80-PerCP5.5 (Biolegend, 400150) and CD86-PE-Cy7 (BD Biosciences, 557872) to assess DC maturation (Violet⁺ population). Isotype controls (PerCP5.5, Biolegend, 305232; PE-Cy7, BD Biosciences, 557872) were included to subtract aspecific signals from measured fluorescence intensity. Phagocytosis of NSCLC cells was assessed by gating on the PKH67⁺Violet⁺ population, as previously described [35 (link)]. Acquisition was performed on a FACSAria II (BD Biosciences). Data analysis was performed using FlowJo v10.1 software (TreeStar).

Cells were incubated for 30 min on ice in 100 μl Hank’s balanced salt solution (Thermo Fisher) + 2% FBS with Fc block (1:100 dilution, clone 2.4G2; BD Biosciences) along with the following antibodies (all from BD Biosciences): CD45 APC-Cy7 (1:500, clone 30-F11), CD11b AF488 (1:500, clone M1/70), CD80 BV421 (1:200, clone 16-1OA1), and CD86 PE-Cy7 (1:500, clone GL1). IA/I-E AF647 (1:500, clone M5/114.15.2; BioLegend). Cells were then fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) according to manufacturer’s instructions. Cells were then incubated with an anti-RVFV antibody (kindly provided by Dr. Robert Tesh and the World Reference Center of Emerging Viruses and Arboviruses) at 1:500 dilution, followed by goat anti-mouse PE secondary antibody (1:1000, Santa Cruz biotechnology). Flow cytometry was performed using a FACSAria Fusion and data were analyzed using FlowJo software. Microglia, other myeloid lineage, and lymphocytes were resolved using CD45 and CD11b expression, with microglia identified as CD45^int CD11b^int, other myeloid as CD45^hi CD11b^hi, and lymphocytes as CD45^hi CD11b^– as previously described [65 (link)].

Prior to flow cytometry, cells were washed in staining buffer (0.05% (w/v) BSA, 2 mM EDTA in 1× PBS) and treated with FcR blocking reagent (Miltenyi Biotec) for 10 min. Subsequently, In vitro differentiated single cell clones and Dox-pDC were stained with the following antibodies for 30 min at 4°C: CD11c-APC, MHC-I-FITC, MHC-II-PE, SiglecH-PE, CD86-PE-Cy7, CD289 (TLR9)-FITC, CD11b-V500, B220-PerCP, CD8α-APC-Cy7 (all BD Biosciences) and CD9-FITC (Thermo Fisher). T lymphocytes were stained with the following antibodies: CD3-FITC, CD4-V500, CD8α-APC-Cy7, CD44-APC and IFNγ-APC-Cy7 (all BD Biosciences); CD62L-PerCP-Cy5.5 and RORγt-PerCP-ef710 (all Thermo Fisher Scientific). Flow cytometry was performed using LSRII and FlowJo analysis software (V10; FlowJo, Ashland, USA).

Nonexpanded donor-matched cells before and after use of the STEM device (n = 6) were stained using antibodies for MSCs and macrophage markers, including CD90-Alexa700 (AbD Serotec), CD45-V450, CD14-FITIC, CD16-BV786, HLA-DR-PE, M2 marker CD206-APC, and M1 marker CD86-PECy7 (all from BD Biosciences).^{14 (link)}
Isotype controls were used for each antibody with nonspecific antibody binding blockade using phosphate-buffered saline containing 10% mouse serum and 1% human IgG. For all flow cytometry analyses, gating was first established (at 1% positivity) on the isotype controls and then applied to the corresponding markers. Data were acquired and analyzed using Cytoflex S (Beckham Coulter).

Lung tissues were minced and digested in Waymouths media (Invitrogen, Carlsbad, CA, USA) containing 25 mg/mL collagenase A (Roche Diagnostics, Mannheim, Germany), 2.5 mg/mL of DNase I (Sigma-Aldrich, St. Louis, MO, USA), 25 mM Hepes (Sigma-Aldrich), and 10% heat-inactivated fetal bovine serum (Invitrogen) for 15 minutes at 37°C and 5% CO₂. Cells were incubated with an Fc-receptor blocker (553141; BD Pharmingen) for 15 minutes on ice and stained with the following antibodies: CD45-V450 (1:150, 560541; BD Biosciences, North Ryde, NSW, Australia), F4/80-PE (1:100, 12-4321-82; eBioscience, San Diego, CA, USA), CD86-PE Cy-7 (1:200, 560501; BD Biosciences, San Jose, CA, USA), and CD206-Alexa Fluor 647 (1:200, 12310; Australian Biosearch, Karrinyup, WA, Australia). Data collected were analysed by using FlowJo cytometric analysis software (Tree Star, Ashland, OR, USA). For gating strategy, see Figure S2 in Additional file
1 in the online data supplement.