For the phenotype analysis of Raw264.7 macrophages, cells were first treated as indicated. Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C. For the phenotype analysis of THP1 macrophages, cells were treated as indicated, harvested and incubated with APC-conjugated F4/80 antibody (17-4801-82, Invitrogen) and PE-conjugated CD163 antibody (12-1639-42, eBioscience) for 30 min at 37 °C. For the phenotypic analyses of primary macrophages isolated from mouse 4 T1-Luc xenografts, cells were incubated with CD45-PE-Cy7 (25-0451-82, eBioscience), F4/80-APC antibody (17-4801-82, Invitrogen), and CD206-PE antibody (141,705, Biolegend) for 30 min at 37 °C. For PD-L1 expression analyses of primary macrophages, cells were incubated with CD45-PE-Cy7 (25-0451-82, eBioscience), F4/80-FITC antibody (SC-71085, Santa Cruz), and PD-L1-APC antibody (124,312, Biolegend) for 30 min at 37 °C. After incubation, the cells were washed once with PBS and subjected to flow cytometry analysis.
Cd45 pe cy7
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD45-PE-Cy7 is a fluorescently labeled antibody that binds to the CD45 protein expressed on the surface of various types of leukocytes. It is a commonly used reagent in flow cytometry applications for the identification and enumeration of different blood cell populations.
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Lab products found in correlation
Cd31 pe cy7, by Thermo Fisher Scientific (16 mentions)
Epcam apc, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Cd4 apc cy7, by BioLegend (4 mentions)
Cd3 fitc, by Thermo Fisher Scientific (4 mentions)
Cd11b percp cy5, by Thermo Fisher Scientific (4 mentions)
C kit apc, by Thermo Fisher Scientific (3 mentions)
Rlt lysis buffer, by Qiagen (3 mentions)
Cd24 apc, by Thermo Fisher Scientific (3 mentions)
Ter119 pecy7, by Thermo Fisher Scientific (3 mentions)
Collagenase, by Merck Group (3 mentions)
F4 80 apc cy7, by BioLegend (3 mentions)
F4 80 apc, by Thermo Fisher Scientific (3 mentions)
Sytox blue, by Thermo Fisher Scientific (3 mentions)
Cd206 alex647, by Bio-Rad (3 mentions)
Facsaria cell sorter, by BD (3 mentions)
Cd11c pe, by BD (3 mentions)
Pdpn efluor660, by Thermo Fisher Scientific (3 mentions)
Facsaria 2, by BD (3 mentions)
Gallios flow cytometer, by Beckman Coulter (3 mentions)
52 protocols using cd45 pe cy7
1
Macrophage Phenotyping by Flow Cytometry
2
Isolation and Characterization of Tumor-Associated Cells
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Orthotopic 4T1 tumours or lungs of 4T1-tumour-bearing mice were isolated. Disassociation of tumours was performed as described21 . For dissociation of lung tissue, lungs were minced into small pieces and incubated for 45 min with DMEM supplemented with 0.1% collagenase II (Worthington, LS4176) and 0.1% Dispase II (Sigma-Aldrich, 4942078001). Single cell suspensions were incubated with anti-mouse CD16/CD32 (eBioscience, 16-0161-82, diluted 1:50) for 15 min to reduce non-specific staining, followed by staining with the following anti-mouse antibodies: CD31 (PECAM-1)-FITC (eBioscience,11-0311-82, diluted 1:50), CD45-PE/Cy7 (eBioscience, 25-0451-82, diluted 1:200), and EpCAM-APC (eBioscience, 17-5791-80, diluted 1:100). ECs were isolated as CD31+CD45−EpCAM− cells. For myeloid cell isolation, tissues were disassociated into single cells suspensions as described above, and stained with the following anti-mouse antibodies: CD45-PE/Cy7 (eBioscience, 25-0451-82, diluted 1:200), CD11b-PerCP/Cy5.5 (eBioscience; 45-0112-82, diluted 1:100), Ly-6G-APC (Biolegend, 127614, diluted 1:200) Ly-6C-FITC (Biolegend, 128006, diluted 1:200). DAPI was used to exclude dead cells. Sorting was performed using BD FACS Aria II.
3
Isolation of Epithelial Cell Subsets
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Adult epithelial cells were collected as described previously (Frank et al. 2016 (link)). NANCIcreERT2-TdTomato and Nkx2.1GFP/+ adult lungs were harvested and processed into single-cell suspensions using a dispase (Collaborative Biosciences)/collagenase (Life Technologies)/DNase solution. Epithelial cells were sorted from the single-cell suspension using either a MoFlo Astrios EQ (Beckman Coulter) or FACSJazz (BD Biosciences) flow cytometer with antibody staining for CD31-PECy7 (eBioscience), CD45-PECy7 (eBioscience), and EpCAM-APC (eBioscience). Following negative selection for CD31 and CD45, epithelial cells were isolated by positive selection for EpCAM, separated based on RFP or GFP expression, and sorted into Trizol LS.
AT2-enriched and AT2-depleted populations were isolated from 18-wk-old mice using a modified version of previously published methods (Alder et al. 2015 (link)). In short, pulmonary cells were digested and sorted as described above using antibody staining for Sca-1-APC (eBioscience), CD34-APC (BioLegend), Pdpn-eFluor660 (eBioscience), CD31-PECy7 (eBioscience), CD45-PECy7 (eBioscience), and EpCAM-eFluor780 (eBioscience). AT2-enriched (PECy7−, eFluor780+, and APC/eFluor660−) and AT2-depleted (PECy7−, eFluor780+, and APC/eFluor660+) cells were sorted into Trizol LS.
AT2-enriched and AT2-depleted populations were isolated from 18-wk-old mice using a modified version of previously published methods (Alder et al. 2015 (link)). In short, pulmonary cells were digested and sorted as described above using antibody staining for Sca-1-APC (eBioscience), CD34-APC (BioLegend), Pdpn-eFluor660 (eBioscience), CD31-PECy7 (eBioscience), CD45-PECy7 (eBioscience), and EpCAM-eFluor780 (eBioscience). AT2-enriched (PECy7−, eFluor780+, and APC/eFluor660−) and AT2-depleted (PECy7−, eFluor780+, and APC/eFluor660+) cells were sorted into Trizol LS.
4
Bronchoalveolar Lavage Fluid Analysis
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Twenty-four hours after the final treatment was administered, the mice were sacrificed, and the BALF was collected by flushing the lungs three times with 0.5 mL of PBS through an intravenous catheter. The total number of cells in the BALF was counted with an automatic cell counter. In addition, the numbers of lymphocytes, eosinophils, and neutrophils were counted by flow cytometry. Specifically, the expression of cell surface markers was assessed using the following fluorescent dye-conjugated mouse antibodies: PE-Cy7-CD45 (eBioscience, San Diego, CA, USA), Alexa 647-F4/80 (BD Biosciences, Sparks, MD, USA), PE-siglecF (BD Biosciences, Sparks, MD), and FITC-Ly6G (eBioscience, San Diego, CA, USA). The data were collected using a FACSCalibur flow cytometer (BD Biosciences, Sparks, MD, USA).
5
Isolation of Muscle Stem Cells
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MuSCs were FACS-sorted from hind limb muscles in postnatal 1-week mice according to the reported protocol [45 (link)]. Briefly, mechanical and enzymatic dissociation of cells, released resident mononucleated cells, then followed by antibody staining and FACS isolation.
Mononuclear cells were incubated with antibodies against CD45 (PE/Cy7-CD45, eBioscience, 25-0451-82), CD31 (FITC-CD31, BioLegend, 102506), Sca1 (V450-Sca1, BD, 560653) and VCAM1 (efluor660-VCAM1, eBioscience, 50-1061-80). Immunostained cells were briefly washed, passed through a 70-μM nylon mesh (Falcon) and suspended at a concentration of 106–107 cells per mL. Cells were further separated with the Beckman Coulter MoFlo XDP system. Sorting gates were strictly defined on the basis of control cells stained with single antibodies as well as the patterns of forward scatter and side scatter of MuSCs in preliminary tests. Collection of the cells that were positive for VCAM1 expression and negative for CD31, CD45, and Sca1.
Mononuclear cells were incubated with antibodies against CD45 (PE/Cy7-CD45, eBioscience, 25-0451-82), CD31 (FITC-CD31, BioLegend, 102506), Sca1 (V450-Sca1, BD, 560653) and VCAM1 (efluor660-VCAM1, eBioscience, 50-1061-80). Immunostained cells were briefly washed, passed through a 70-μM nylon mesh (Falcon) and suspended at a concentration of 106–107 cells per mL. Cells were further separated with the Beckman Coulter MoFlo XDP system. Sorting gates were strictly defined on the basis of control cells stained with single antibodies as well as the patterns of forward scatter and side scatter of MuSCs in preliminary tests. Collection of the cells that were positive for VCAM1 expression and negative for CD31, CD45, and Sca1.
6
Multiparameter Flow Cytometry Immunophenotyping
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The surface markers included FITC‐CD3 (MA1‐7640, eBioscience, San Diego, CA, USA), APC‐Cy7‐CD4 (100413, Biolegend, San Diego, CA, USA), APC‐CD4 (100411, Biolegend), PerCP‐CD8 (100,731, Biolegend), PE‐Cy7‐CD8 (100721, Biolegend), PE‐Foxp3 (12‐5773‐82, eBioscience), APC‐Helios (17‐9883‐42, eBioscience), PerCP‐CD44 (103035, Biolegend), FITC‐CD62L (104405, Biolegend), FITC‐lin (22‐7770‐72, eBioscience), PerCP‐CD34 (50‐0341‐82, eBioscience), APC‐Sca‐1 (17‐5981‐82, eBioscience), PE‐Sca‐1 (12‐5981‐82, eBioscience), APC‐c‐kit (17‐1171‐82, eBioscience), PE‐c‐kit (12‐1171‐82, eBioscience), PE‐B220 (12‐0452‐82, eBioscience), FITC‐IgM (11‐5790‐81, eBioscience), PE‐Cy7‐CD19 (12‐0193‐82, eBioscience), APC‐CD19 (17‐0193‐82, eBioscience), PE‐Cy7‐CD16/32 (25‐0161‐82, eBioscience), PE‐Cy7‐CD45 (25‐0451‐82, eBioscience), PerCP‐5.5‐CD45.2 (45‐0454‐82, eBioscience), and APC‐Cy7‐CD45.1 (17‐0453‐82, eBioscience) obtained from Invitrogen. Intracellular staining with PE‐Foxp3 (72‐5775‐40, eBioscience) was performed with Foxp3 staining kits (Invitrogen, Carlsbad, CA, USA).
7
Isolation of Skeletal Muscle Stem Cells
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MuSCs were FACS-sorted from hind limb muscles of postnatal 1-week mice as previously described (Xie et al., 2018 (link)). Briefly, mechanical and enzymatic dissociation of cells, released resident mononucleated cells, then followed by antibody CD45 (PE/Cy7-CD45, eBioscience, 25-0451-82), CD31 (FITC-CD31, BioLegend, 102506), Sca1 (V450-Sca1, BD, 560653) and VCAM1 (efluor660-VCAM1, eBioscience, 50-1061-80) staining and FACS isolation. Collection of the cells those were positive for VCAM1 expression and negative for CD31, CD45, and Sca1.
8
Single-Cell Sorting of Activated B Cells
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Within 5 h after collection, tissues were minced and digested with Liberase TL (Sigma-Aldrich, 5401020001) for 15 min at 37 °C. Cells were washed and stained for 30 min at 4 °C with Calcein AM (ThermoFisher Scientific, 65-0853-78) and antibodies: PE-CD19 (ThermoFisher Scientific, clone: SJ25C1, 12-0198-42), APC-CD38 (BD Biosciences, clone: HIT2, 555462), PE-Cy7-CD45 (ThermoFisher Scientific, clone: HI30, 25-0459-42). Stained cells were washed, and DAPI (Thermo Fisher Scientific, D1306) was added to the single-cell suspension immediately before the samples were subjected to BD FACSAria Fusion with FACSDiva 8.0.1 for sorting. Doublets were excluded by FSC-A/FSC-H gating, and CD45 + Calcein+ DAPI- CD19 + CD38 + activated B cells were single-cell sorted into 96-well plates with catching buffer (RLT lysis buffer (Qiagen, 79216) with 1% 2-mercaptoethanol (Sigma-Aldrich, 63689-25ML-F)). Sorted cells were immediately spun down and stored at -80 °C until being processed for scRNA-seq. Data were analyzed with FlowJo 10.7.1.
9
Multi-Marker Flow Cytometry Protocol
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FITC- and APC-CD19, CD11b, GR-1, FITC-TER119, FITC-CD44, PE-Dll1, Dll4, APC-c-kit, CD11c, B220, ST2, PE/Cy7-CD4, APC/Cy7-Thy1.2 and CD19 mAbs were all purchased from BioLegend (San Diego, CA). PE-CD19, PerCP/Cy5.5-CD25, APC-CD8, DX5, PDGFRα and PE/Cy7-CD45 mAbs were purchased from Thermo Fisher Scientific (Tokyo, Japan). Anti-NotchLs mAb, HRJ1-5, was established by immunization of Armenian hamsters with a recombinant rat Jagged1-human Fc chimeric protein (34) (R and D Systems Inc, Minneapolis, MN). Expression of NotchLs assessed by HRJ1-5 was detected by biotinylated goat anti-hamster IgG Ab (Thermo Fisher Scientific) and PE-conjugated streptavidin (BD Bioscience). Intracellular staining of HA-labeled Dll1 or Dll4 was detected by rabbit anti-HA mAb (Cell Signaling Technology) and DyLight649-conjugated donkey anti-rabbit IgG Ab (BioLegend). Expression of cell surface markers was analyzed by flow cytometry with a FACSVerse (BD Biosciences).
10
Isolation and Characterization of Mouse Skin Cells
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Intact dorsal mouse skin or wound beds were harvested, minced, and digested using Liberase TM to release single cells. Cells were resuspended in FACS staining buffer (1% BSA in PBS with 2 mM EDTA) and stained with the following antibodies for 30 min on ice: CD45-PE-Cy7 (eBioscience, 1:2000), CD31-APC-Fire750 (Biolegend 1:1000), CD29-Alexa700 (eBioscience, 1:400), CD34-Pacific Blue (BD Bioscience, 1:50), Sca1-V500 (eBioscience, 1:500), CD26-PE-Cy7 (Biolegend, 1:500), CD9-APC (BD Bioscience, 1:100), and CD9-FITC (eBioscience, 1:100). Samples were analyzed on a BD Aria III with FACSDiva software (BD Biosciences, San Jose, CA, USA), and gating and analyses were completed with FlowJo software (BD Biosciences).
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