To highlight HBsAg uptake/binding on DC subsets, PBMCs were seeded at 1 × 106 cells mL−1 in 48‐well plates (Corning) and exposed for 2 h at 4 or 37°C to 25 µg mL−1 of either native or deglycosylated FL‐HBsAg or FL‐HBcAg in presence or not of a mixture of TLR ligands comprising polyI:C (30 µg mL−1), Imiquimod (R848, 1 µg mL−1), and Class‐A CpG oligonucleotide ODN‐2336 (CpGA, 1 µm ; Invivogen). In some experiments, PBMCs were exposed to increased amounts of FL‐HBsAg from 10 to 75 µg mL−1. After exposure, PBMCs were thoroughly washed with cold PBS2% FBS to remove the unbound particles and cells were then stained for CD11c, HLA‐DR, Lineage cocktail, CLEC9A, CD123 (BD); CD45 (Biolegend), and BDCA1 (Beckman Coulter) antibodies and further fixed with FACS lysing solution (BD). FL‐HBsAg‐positive and FL‐HBcAg‐positive cDC1s, cDC2s and pDCs were then analysed using LSRII Flow Cytometer and FACSDiva software (BD).
Lineage cocktail
Manufactured by BD
The Lineage cocktail is a laboratory reagent used for cell culture applications. It serves as a growth medium for the maintenance and expansion of cell lines. The cocktail provides a balanced combination of nutrients, growth factors, and other essential components required for the optimal proliferation and viability of cultured cells.
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Lab products found in correlation
Cd123, by BD (4 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Bdca1, by Beckman Coulter (2 mentions)
Facsdiva software, by BD (2 mentions)
Facs lysing solution, by BD (2 mentions)
48 well plate, by Corning (2 mentions)
Ckit pe cy7, by BioLegend (2 mentions)
Cd4 apc h7, by BD (2 mentions)
Cd45 2 percp cy5, by BD (2 mentions)
Cd48 apc cy7, by BD (2 mentions)
Cd127 ecd, by BD (2 mentions)
Gr1 apc alexa700, by BD (2 mentions)
Sca1 brilliant violet 421, by BioLegend (2 mentions)
Aquavivid, by Thermo Fisher Scientific (2 mentions)
Cd150 percp cy5, by BioLegend (2 mentions)
Cd11b pe, by BD (2 mentions)
Cd3 pe cy7, by BD (2 mentions)
Cd8 ecd, by Beckman Coulter (2 mentions)
Facscalibur, by BD (2 mentions)
Cd24 pe cy7, by BD (2 mentions)
16 protocols using lineage cocktail
1
Quantifying HBsAg Uptake in Dendritic Cell Subsets
2
Murine Hematopoietic Cell Immunophenotyping
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Cells were isolated from bone marrow, spleen, and thymus of mice, and stained with the following antibodies: B220 (APC; BD Pharmingen), cKit (PE-Cy7; BioLegend, San Diego, CA), CD3 (PE-Cy7; BD Bioscience), CD4 (APC-H7; BD Pharmingen), CD8 (ECD; Beckman Coulter), CD11b (PE; BD Bioscience), CD16/32 (PE; eBioscience, San Diego, CA), CD34 (FITC; BD Pharmingen), CD45.2 (PerCP-Cy5.5; BD Pharmingen), CD48 (APC-Cy7; BD Pharmingen), CD127 (ECD; BD Pharmingen), CD150 (PerCP-Cy5.5; BioLegend), Gr1 (APC-Alexa700; BD Bioscience), Lineage Cocktail (APC; BD Pharmingen), Sca1 (Brilliant Violet 421; BioLegend). Dead cells were excluded using either DAPI or Vivid-Aqua (Invitrogen) staining. All data acquisition was performed on a LSRII (BD) flow cytometer, and results were analyzed using FlowJo v.8.8.7 (TreeStar).
3
Neutralizing CLR Binding of HBsAg on DCs
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To determine the involvement of specific CLRs as putative attachment factors for HBsAg on DCs, we performed neutralisation experiments by blocking single CLR using specific functional grade anti‐human CLR antibodies. Briefly, PBMCs were seeded at 2 × 106 cells mL−1 in 48‐well plates (Corning) and pre‐incubated for 30 min at 37°C in presence or not of blocking anti‐human DCIR, FcɣRIIA, MMR, FcεRIα, DECTIN1, BDCA2 (R&D systems), NKp44 (Invitrogen and Biolegend), ILT7 (Invitrogen) antibodies (single or in combination) or specific isotype controls. After 30 min of CLR blockade, 25 µg mL−1 of FL‐HBsAg was added in the culture for an additional 2 h at 37°C. Subsequently, PBMCs were thoroughly washed with cold PBS2% FBS to remove solution‐free anti‐human CLR antibodies or FL‐HBsAg. Hence, cells were stained with anti‐CD11c, HLA‐DR, Lineage cocktail, CLEC9A, CD123, (BD), CD45 (Biolegend) and BDCA1 (Beckman Coulter) antibodies and fixed with FACS lysing solution (BD). FL‐HBsAg‐positive cDC1s, cDC2s and pDCs in each condition were then analysed using LSRII Flow Cytometer and FACSDiva software (BD).
4
Murine Bone Marrow Cell Phenotyping
5
Multiparametric Flow Cytometric Analysis of HSCs
6
Multiparametric flow cytometry analysis
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Single-cell suspensions of BM and skin-infiltrating leukocytes were incubated at 4 °C for 20 min in staining buffer (1 × PBS with 0.1% bovine serum albumin and 0.1% sodium azide) containing the appropriate antibody mix. NOS2-AF488, CD45.1-eFluor 450, Ly6G-FITC, Ly6G-PE, Ly6G-PE.Cy7, CD11b-PE, CD11b-APC, F4/80-PE, CD115-APC, Ly6C-APC, CD45-PE, B220-PE, CD4-APC, CD8-APC, and CD105-APC antibodies were obtained from eBioscience. Lineage cocktail-APC, CD11c-APC.Cy7, α-BrdU-FITC and CD34-PE antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Arginase-FITC antibody was purchased from R&D systems (Minneapolis, MN, USA). For the flow cytometric analysis of Lineage-positive cells, the cells were stained with biotinylated Lineage cocktail obtained from BD Pharmingen, then labeled with streptavidin-V450 (BD Pharmingen). Data were collected using a FACSCalibur or LSRII-Green (both BD Pharmingen) and were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).
7
Comprehensive Multicolor Flow Cytometry
8
Isolation and Phenotyping of Lung Macrophages
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Lung single-cell suspensions were blocked with anti-mouse CD16/32 (BD Bioscience) and then, stained with fluorochrome-conjugated antibodies against CD45, F4/80, CD11b, CD86, CD206, and ST2 (BD Bioscience). Lineage cocktail included antibodies to CD3, CD4, CD5, CD8, CD11b, Gr-1, CD19, B220, DX5, and TCRδ (BD Bioscience). To obtain lung macrophages, single-cell suspensions from the lungs of tested mice were separated by magnetic bead purification of F4/80 positive cells (Invitrogen, Thermo Fisher Scientific, CA 8802–6863).
9
Phenotypic Analysis of Single Cells
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Single cells were labeled with antibodies for CD24-PeCy7 and CD29-PacBlue, or Annexin V-PacBlue (BD Pharmingen), based on the manufacturer’s data sheet. Lineage cells were excluded using the BD Pharmingen lineage cocktail. Dead cells were excluded by staining with Sytox Red (1:1000).
10
Phenotypic Analysis of Single Cells
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