A nonparenchymal cell fraction from whole liver extracts was isolated upon collagenase and mechanical digestion followed by Percoll (GE Healthcare Life Sciences) gradient centrifugation as previously described [5 (link)]. In parallel, blood samples were collected in EDTA-containing tubes and treated with red blood cell lysis buffer (PharmLyse, BD Biosciences, Germany). Upon removal of red bodies and centrifugation, immune cells were incubated with fluorochrome-conjugated antibodies and characterized according to two different panels, a myeloid panel: CD45-BV510 (103138, BioLegend), 7AAD-PE-Cy5-YG (420404, BioLegend), CD11b-BV711 (101242, BioLegend), F4/80-APC (17-4801-82, eBiosciences), MHC2-Alexa700 (107622, BioLegend), CD11c-PE-Cy7 (25-0114-81, eBiosciences), and Ly6G-FITC (551460, BD Pharmingen) and a lymphoid panel: CD45-BV510 (103138, BioLegend), 7AAD-PE-Cy5-YG (420404, BioLegend), CD3-PE-Cy7 (25-0031-82, eBiosciences), CD4-FITC (11-0041-85, eBiosciences), CD8-PerCpCy5.5 (126610, BioLegend), and NK1.1-BV711 (108745, BioLegend). Labeled cells were then subjected to flow cytometry using a BD Canto II (BD Biosciences) and relative cell numbers were analyzed using FlowJo software (Tree Star).
Cd45 bv510
Manufactured by BioLegend
Sourced in United States
CD45-BV510 is a fluorophore-conjugated antibody that binds to the CD45 antigen, which is expressed on the surface of hematopoietic cells. This product can be used in flow cytometry applications for the identification and analysis of different immune cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 percp cy5, by BioLegend (4 mentions)
Precision count beads, by BioLegend (4 mentions)
Fc block, by BD (4 mentions)
Cd4 fitc, by Thermo Fisher Scientific (3 mentions)
Cd4 bv421, by BioLegend (3 mentions)
Ly6g bv421, by BioLegend (3 mentions)
Ly6g pe, by BioLegend (3 mentions)
Cd3 fitc, by BioLegend (3 mentions)
Lsrfortessa, by BD (3 mentions)
Cd8 bv785, by BioLegend (3 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (3 mentions)
Cd4 fitc, by BioLegend (3 mentions)
Pharm lyse buffer, by BD (3 mentions)
F4 80 apc, by Thermo Fisher Scientific (2 mentions)
Cd11c pe cy7, by Thermo Fisher Scientific (2 mentions)
Ly6g fitc, by BD (2 mentions)
Cd19 pe cy7, by BioLegend (2 mentions)
Fcr blocking reagent, by Miltenyi Biotec (2 mentions)
Epcam fitc 9c4, by BioLegend (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
39 protocols using cd45 bv510
1
Multiparametric Analysis of Liver-Derived Immune Cells
2
Single-cell Analysis of Intestinal Immune Cells
3
Isolation of Murine and Human Intestinal Cells
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After 4 days of murine and 7 days of human co-culture, Matrigel was disrupted and cells were collected into 15ml falcon tubes. For murine ILC1 cultures Matrigel disruption was not required, and cells were gently rinsed from the bottom of the plate using PBS+2%FCS. Samples were rinsed with PBS, then dissociated in TrypLe (Gibco) for 20mins at 37°C. The sorting buffer after this step contained DNAse (250µg/ml), EDTA (1μl/ml), and HEPES (1μl/ml) to maintain single epithelial cells and avoid clumping. Cells were titruated gently, centrifuged and resuspended in sorting buffer. Cells were then filtered (70µm), having pre-coated the filter with sorting buffer to minimize cell loss, and either rinsed with PBS for fixable Live/Dead staining (UV or nearInfraRed, Thermofisher), or stained with EpCAM, CD45, and the requisite combination of antibodies for the experiment, and analyzed (BD Fortessa) or sorted (BD ARIA3 Fusion & BD Aria 2 using BD FACS Diva 8.0.1 software). Isolation of murine IEC and ILC1 following co-culture was performed using EpCAM–APC Cy7 (G8.8, BioLegend), CD45-BV510 (30-F11, bioLegend), NK1.1 BV605 (PK136, BioLegend), CD44-PE (IM7, BioLegend). Isolation of human IEC, FB, and hILC1 used fixable Live/Dead-UV or Live/Dead-nIR CD45-eFluor450(HI30) Invitrogen, EpCAM-FITC (9C4; BioLegend), CD90-PE/Dazzle (Thy1; BioLegend)
4
Immunophenotyping of Tumor Cells and CAR T Cells
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The following Abs were purchased from BD bioscience: human CD3-APC-H7 (Cat#: 560176, dilution: 1:100), CD4-BV421 (Cat#: 562424, dilution: 1:100), CD8-APC (Cat#: 340584, dilution: 1:100), CD45-BV510 (Cat#: 563204, dilution: 1:30), CD45RA-PE (Cat#: 555489, dilution: 1:30), CCR7-FITC (Cat#: 561271, dilution: 1:50) and B7-H3-BV421 (Cat#: 565829, dilution: 1:100); human CCR2-BV421 (Cat#: 357210, dilution: 1:100) Ab was purchased from Biolegend. Expression of human B7-H3 in tumor cell lines and organoid cells was assessed with the 376.96 mAb followed by APC-goat anti-mouse IgG Ab (BD Biosciences, Cat# 550826, RRID: AB_398465, dilution 1:100), and confirmed with another B7-H3 mAb (Clone 7–517; BD Bioscience, Cat#: 565829, RRID: AB_2739369, dilution 1:100)32 (link). Expression of the B7-H3.CARs was detected using the fusion protein 2Ig-B7-H3-Fc (R&D Cat# 1027-B3) and followed by AF647-goat anti-human IgG (H+L) Ab (Jackson ImmunoResearch Laboratories Inc., Cat# 109-606-088, dilution 1:100); CD19.CAR was detected with the previously described anti-CD19.CAR mAb47 (link). Samples were acquired with BD FACS Canto II or BD FACS Fortessa using the BD Diva software (BD Biosciences). For each sample, a minimum of 10,000 events were acquired and data were analyzed using Flowjo 10.
5
Inflammasome Activation Assay Protocol
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HDM was from Greer Laboratories. Anti-mouse IL-1β (p17) (AF-401-NA) was from R&D. Anti-mouse caspase-1 (p20) (AG-20B-0042) and anti-NLRP3 (AG-20B-0014) antibodies were from Adipogen. Anti-ASC (67824) antibody was from Cell Signaling Technology. Anti-β-actin (66009-1-Ig) was from Proteintech.
Anti-mouse antibodies used for flow cytometry were: CD3-FITC (BD, 553062, 145-2C11), CD19-FITC (eBioscience, 11-0193-82, 1D3), Ly-6G-PE (Biolegend, 127608, 1A8), CD45-PE (eBioscience, 12-0451-81, 30-F11), CD11c-PerCP-Cy5.5 (Biolegend, 117328, N418), CD11b-PerCP-Cy5.5 (BD, 550993, M1/70), CD11b-PE-Cy7 (eBioscience, 25-0112-82, M1/70), CD3e-PE-Cy7 (BD, 552774, 145-2C11), CD19-PE-Cy7 (Biolegend, 115520, 6D5), SiglecF-Alexa Fluor 647 (BD, 562680, E50-2440), MHCII-APC-eFluor 780 (eBioscience, 47-5321-82, M5/114.15.2), Ly-6G-BV421 (Biolegend, 127628, 1A8), CD45-BV510 (Biolegend, 103137, 30-F11), CD11c-BV510 (Biolegend, 117338, N418). Ultrapure LPS was obtained from Invitrogen. Nigericin was obtained from Sigma-Aldrich. RRx-001 (S8405) was from Selleck.
Anti-mouse antibodies used for flow cytometry were: CD3-FITC (BD, 553062, 145-2C11), CD19-FITC (eBioscience, 11-0193-82, 1D3), Ly-6G-PE (Biolegend, 127608, 1A8), CD45-PE (eBioscience, 12-0451-81, 30-F11), CD11c-PerCP-Cy5.5 (Biolegend, 117328, N418), CD11b-PerCP-Cy5.5 (BD, 550993, M1/70), CD11b-PE-Cy7 (eBioscience, 25-0112-82, M1/70), CD3e-PE-Cy7 (BD, 552774, 145-2C11), CD19-PE-Cy7 (Biolegend, 115520, 6D5), SiglecF-Alexa Fluor 647 (BD, 562680, E50-2440), MHCII-APC-eFluor 780 (eBioscience, 47-5321-82, M5/114.15.2), Ly-6G-BV421 (Biolegend, 127628, 1A8), CD45-BV510 (Biolegend, 103137, 30-F11), CD11c-BV510 (Biolegend, 117338, N418). Ultrapure LPS was obtained from Invitrogen. Nigericin was obtained from Sigma-Aldrich. RRx-001 (S8405) was from Selleck.
6
Multiparametric Flow Cytometry Protocol
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Single-cell suspensions were incubated in anti-CD16/CD32 Fc block antibody (5 μg/ml; clone 2.4G2; cat # 553141, BD Biosciences, San Jose, CA) and fixable viability dye eFluor 780 (Invitrogen, Carlsbad, CA) for 15 min at 4 °C in cold PBS. Cells were then washed once and stained with combinations of the following antibodies: CD45-BV510 (2 μg/ml; clone 30-F11; cat # 103138, BioLegend, San Diego, CA), TCRβ-PerCP-Cy5.5 (2 μg/ml; clone H57-597; cat # 109228, BioLegend, San Diego, CA), CD4-BUV395 (2 μg/ml; clone GK1.5; cat # 563790, BD Biosciences, San Jose, CA), CD8-af700 (5 μg/ml; clone KT15; cat # MCA609A700, Bio-Rad, Hercules, CA), NKp46-APC (1 μg/ml; clone 29A1.4; cat # 137607, BioLegend, San Diego, CA), CD49b-PE (2 μg/ml; clone DX5; cat # 108908, BioLegend, San Diego, CA), B220-BUV661 (2 μg/ml; clone RA3-6B2; cat # 612972, BD Biosciences, San Jose, CA), and Thy1.2-BUV805 (1 μg/ml; clone 53-2.1; cat # 741908, BD Biosciences, San Jose, CA). Data were acquired on a Symphony flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (FlowJo LLC, Ashland, OR). Gating strategies for the identification of CD8+ T cells and NK cells are provided in Supplementary Fig. 13 .
7
Comprehensive Immune Cell Analysis
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The following antibodies were used to characterize BMDCs and assess their activation state: MHC II-PE, CD11c-APC, CD86-PE-Cy7, CD80-FITC and CD11b-APC-Cy7 (all from BioLegend). Acquisition was performed on an Attune NxT (ThermoFisher). Leukocyte populations from BM, liver and spleen were also analyzed by flow cytometry. Single cells were excluded from dead cells using the LIVE/ DEAD Zombie NIR Fixable Viability Kit (BioLegend). Immuno-phenotyping was performed using the following antibodies: CD45-BV510, CD49b-PE-Dazzle, CD19-PerCP, CD3-FITC, CD4-AF700, CD8-BV785, C44-BV650 and CD62L-BV421 (all from BioLegend). Full minus one (FMO) controls were used to determine positivity. Precision count beads (BioLegend) were used to count immune cells in different organs. Before acquisition, stained cells were fixed with 1% Paraformaldehyde (Sigma-Aldrich). Acquisition was performed using the BD LSRFortessa and data were analyzed using the FlowJo software (TreeStar) version 10.
8
Isolation of Murine and Human Intestinal Cells
9
Immunophenotyping of Lymphocytes
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Cell staining, flow cytometry and intracellular staining was performed as previously described. [26 (link)] Isolated spleen and liver infiltrating lymphocytes were stained with Live Dead Near-IR Dead Cell kit (Invitrogen), CD3-BV785 (Biolegend), CD45-BV510 (Biolegend), CD8-PE-Cy7 (Biolegend), CD4-PE-CF594 (Becton Dickinson), and CD25-PE-Cy7 (Becton Dickinson). Intracellular staining was performed using anti-mouse forkhead box P3 (FoxP3)-AF488 (MF23; BD Pharmingen). Intracellular cell staining for IFNγ and flow cytometry was performed as previously described using IFNγ-BV421 (Biolegend) [26 (link)].
10
Isolation and Characterization of Murine Adipose SVCs
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Epididymal fat pads from db/m or db/db mice were minced and digested in PBS buffer containing 1 mg/ml type II collagenase (Sigma-Aldrich, St. Louis, MO) and 2% BSA (pH 7.4) at 37 °C for 30 min. The cell suspension was filtered through a 100 μm cell strainer and centrifuged at 1500 × g for 10 min. The floating adipocyte fraction was removed from the SVC pellet. The pellet was incubated with RBC lysis buffer (Thermo Fisher Scientific, Waltham, MA) for 5 min, re-centrifuged at 1500 × g for 10 min, and the pellet resuspended in PBS buffer supplemented with 2% FBS. Finally, SVCs were incubated with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4 °C for 20 min, followed by an incubation with fluorochrome-conjugated antibodies to CD45 BV510, CD11b PE, CD11c BV421 (Biolegend, San Diego, CA), or F4/80 APC (Thermo Fisher Scientific, Waltham, MA). Labeled cells were analyzed using FACSAria II (BD Biosciences, NewJersey, USA) and data analysis was done using Flow Jo (Tree Star) (Supplementary Fig. 14 ).
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