B cells were eluted from PBMCs using a MACS Human B Cell isolation kit (Miltenyi Biotec). B cells were stained with rGP38 (IbAr10200) that had been tetramerized at 25 nM using Streptactin-PE (IBA Lifesciences) and Streptactin-APC (IBA Lifesciences). B cells were simultaneously stained with rGP38-Streptactin-PE and rGP38-Streptactin-APC tetramers for 1 hour on ice. Cells were washed twice in buffer (PBS, FBS, EDTA). Next, B cells were stained with a panel of antibodies. Donor 1 PBMCs were stained with a cocktail of anti-human CD3 PerCP-Cy5.5 (Biolegend), CD8 PerCP-Cy5.5 (Biolegend), CD14 PerCP-Cy5.5 (Invitrogen), CD16 PerCP-Cy5.5 (Biolegend), propidium iodide (PI) (Invitrogen), CD19 PE-Cy7 (Biolegend), CD27 BV510 (BD Biosciences), IgM BV711 (BD Biosciences), IgD BV421 (Biolegend), IgG BV605 (BD Biosciences), and IgA AF488 (Abcam). Donor 5 and 6 PBMCs were stained with a cocktail of anti-human CD3 PerCP-Cy5.5 (Biolegend), CD8 PerCP-Cy5.5 (Biolegend), CD14 PerCP-Cy5.5 (Invitrogen), CD16 PerCP-Cy5.5 (Biolegend), PI (Invitrogen), CD19 PE-Cy7 (Biolegend), CD20 PE-Cy7 (Biolegend), CD27 BV510 (BD Biosciences), IgM AF488 (Biolegend), and IgD BV421 (Biolegend). B cells were washed twice in buffer and run on a FACS Aria Fusion Cytometer (BD Biosciences). B cells were sorted into Super Script III reaction buffer (ThermoFisher Scientific) in 96-well Costar plates and frozen at −80 °C.
Cd14 percpcy5
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD14-PerCPCy5.5 is a fluorescently labeled antibody that binds to the CD14 protein, which is expressed on the surface of certain immune cells. It is commonly used in flow cytometry applications to identify and analyze these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 pe cy7, by BD (2 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Anti cd3 pb, by BD (2 mentions)
Anti cd56 apc, by Beckman Coulter (2 mentions)
Qiashredder, by Qiagen (2 mentions)
Facsaria iiiu, by BD (2 mentions)
Cd209 pe, by BD (2 mentions)
Anti cd1c pecy7, by BioLegend (2 mentions)
Anti slan fitc, by Miltenyi Biotec (2 mentions)
Live dead fixable near ir dead cell stain kit, by Miltenyi Biotec (2 mentions)
Cd3 pb, by BD (2 mentions)
Anti cd11c pe, by BD (2 mentions)
Cd11c apc, by BD (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Strep tactin pe, by IBA Lifesciences (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Streptactin apc, by IBA Lifesciences (1 mentions)
Cd19 pe cy7, by BioLegend (1 mentions)
Igm af488, by BioLegend (1 mentions)
Cd20 pe cy7, by BioLegend (1 mentions)
8 protocols using cd14 percpcy5
1
Tetramer-based Identification and Sorting of B Cells
2
Multicolor Flow Cytometry Panel
3
Comprehensive Cervical Cell Phenotyping
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The phenotype of cells collected on the cervical brushes was determined using flow cytometry. Squamous epithelial cells and dead leukocytes absorbed 4′,6-diamino-2-phenylindole (DAPI). Cells that excluded DAPI were examined using directly labelled monoclonal antibodies against the following human proteins: CD45-FITC and CD16-PerCPCy5.5 from BD Biosciences (Franklin Lakes, NJ, USA), CD163-PE, CD11b-PE, CD20-PE, CD14-PerCPCy5.5, HLA-DR-PECy7, CD19-PECy7, CD27-PECy7, CCR7-PECy7, CD11b-APC (activation epitope CBRM1/5) and CD16-APC from eBioscience (San Diego, CA, USA); CD235a-PE, CD68-PE, CD66b-PE, CD103-PE, CD11c-PE, CX3CR1-PE, CCR2-PerCPCy5.5, CD3-PECy7, CCR1-APC, CD4-APC, CD33-APC and CD64-APC from Biolegend (San Diego, CA, USA). Flow cytometric data were collected on an LSRII (BD Biosciences). A minimum of 300,000 events were collected for each group of antibodies so that even a leukocyte population consisting of 0.01% of the total host-derived cells could be reliably detected. Data were analysed using FlowJo 9.6.4 (TreeStar, Ashland, OR, USA).
4
Sorting and Profiling of Immune Cells
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ErcDCs and macrophages were sorted from ccRCC tissue-cell-suspensions stained with CD45-PeCy7, CD11c-APC, CD3-PB, CD209-PE (all BD Biosciences), CD14-PerCPCy5.5 (eBioscience, San Diego, CA, USA) and LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific). Sorting gates were set on CD209+CD14+ cells (ercDCs) and CD209−CD14+ cells (macrophages), among pre-gated CD45+, live, single CD11c+ CD3− cells. CD1c+ DC and slanDCs were sorted from B- and NK-depleted PBMCs of healthy donors (HD) using anti-CD11c-PE, anti-CD3-PB (all BD Biosciences), anti-CD56-APC (Beckman Coulter, Brea, CA, USA), anti-CD19-PB (Dako by Agilent, Santa Clara, CA, USA), anti-CD1c-PeCy7 (Biolegend, San Diego, CA, USA), anti-slan-FITC (Miltenyi Biotec) and LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit. The gating strategy and instrument parameters are in Supplemental Figure S1 . Gates were set very strictly, not covering the whole population, to avoid contamination with other cell populations. Cell population purity varied between 98–100%. Cells were directly sorted into 250 µL of RLT lysis buffer with ß-mercaptoethanol (RNeasy Micro Kit by Qiagen, Venlo, The Netherlands) using FACSAria IIIu (BD Biosciences), then homogenized (QIAshredder by Qiagen) and stored at −80 °C. Details about sorted cell types and numbers of biological replicates are listed in Table S2 .
5
Isolation and Characterization of Kidney Dendritic Cells
6
Multiparametric Flow Cytometry Analysis
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A flow cytometry analysis was performed using a flow cytometer CyFlow (Partec). The cells were washed with FACS buffer (PBS, 2% FBS) and resuspended in a 0.1 mL FACS buffer. The cells were stained with anti-mouse CD105-APC, CD90.2-FITC, CD34-FITC, CD11b-APC (Miltenyi Biotec), CD14 PerCP‐Cy5.5 (eBioscience), Ly-6A/E (Sca-1)-Pacific Blue (BioLegend) antibodies. Data were analyzed using the FlowJo software (BD).
7
Quantifying Antigen-Specific T-Cell Frequencies
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Analysis of T-cell frequency was accomplished using our previously published approach (26 (link)). Briefly, 40–60 × 106 PBMCs were resuspended in a total of 400–600 µL media, divided into two or three independent tubes of 20 × 106 cells (200 µL) each, incubated with 50 nmol/L dasatinib for 10 min at 37°C, and stained with 20 µg/mL PE-labeled, PE-CF594–labeled, or PE-Cy5–labeled HIP tetramers at room temperature for 120 min (three tetramers per tube for a total of six hybrid peptide tetramers plus an nonimmunogenic control tetramer if adequate cells were available for a third staining tube). Cells were washed, incubated with PE-magnetic beads (Miltenyi Biotec) for 20 min at 4°C, and magnetically enriched; 1% of the cells were retained as a nonenriched sample. Enriched (bound) and nonenriched (precolumn) samples were stained with CD4 V500, CD14 PerCP-Cy5.5, and CD19 PerCP-Cy5.5 (eBioscience) and CD45RA AF700 (BD), CXCR3 FITC, CCR6 BV421, and CCR4 BV605 (BioLegend) for 15 min at 4°C. After washing, cells were labeled with ViaProbe (BD Biosciences) and analyzed on a FACSCanto (BD Biosciences), gating on CD4+CD14−CD19−ViaProbe− cells and plotting tetramer versus CD45RA. Frequencies were calculated as previously described (26 (link)).
8
Isolation and Sorting of Immune Cell Subsets
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ErcDCs and macrophages were sorted from ccRCC tissue-cell-suspensions stained with CD45-PeCy7, CD11c-APC, CD3-PB, CD209-PE (all BD Biosciences), CD14-PerCPCy5.5 (eBioscience) and LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific). Sorting gates were set on CD209 + CD14 + cells (ercDCs) and CD209 -CD14 + cells (macrophages), among pre-gated CD45+, live, single CD11c + CD3 -cells. CD1c + DC and slanDCs were sorted from B-and NK-depleted PBMCs of healthy donors (HD) using anti-CD11c-PE, anti-CD3-PB (all BD Biosciences), anti-CD56-APC (Beckman Coulter), anti-CD19-PB (Dako), anti-CD1c-PeCy7 (Biolegend), anti-slan-FITC (Miltenyi Biotec) and LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit.
Gating strategy and instrument parameters are in supplemental Figure S2. Gates were set very strictly, not covering the whole population, to avoid contamination with other cell populations. Cell population purity varied between 98%-100%. Cells were directly sorted into 250 µl of RLT lysis buffer with ß-mercaptoethanol (RNeasy Micro Kit, Qiagen) using FACSAria IIIu (BD Biosciences), then homogenized (QIAshredder, Qiagen) and stored at -80°C. Details about sorted cell types, number of biological replicates are listed in table S2).
Gating strategy and instrument parameters are in supplemental Figure S2. Gates were set very strictly, not covering the whole population, to avoid contamination with other cell populations. Cell population purity varied between 98%-100%. Cells were directly sorted into 250 µl of RLT lysis buffer with ß-mercaptoethanol (RNeasy Micro Kit, Qiagen) using FACSAria IIIu (BD Biosciences), then homogenized (QIAshredder, Qiagen) and stored at -80°C. Details about sorted cell types, number of biological replicates are listed in table S2).
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