Tibias were collected in sterile Ca2+ and Mg2+ free PBS, crushed, and digested in collagenase A (Sigma-Aldrich, #SCR136) for 30 min at 37 °C to obtain a single-cell suspension. Equal number of cells were immunostained with Endomucin antibody (Santa Cruz, #sc-65495) for 45 min at 4 °C. After washing, cells were immunostained with PE-CD45 (BD Pharmingen, #553081), APC-Ter119 (BD Pharmingen, #557909), PE-Cy7-CD31 (BD Pharmingen, #561410), and Alexa Fluor 647 goat anti-rat secondary antibody (Thermo Fisher, #A-21247) for 45 min at 4 °C, followed by DAPI staining for 5 min before analysis and sorting. Finally, cells were acquired on a BD FACS Verse flow cytometer and sorted on a FACS Aria II flow cytometer. To demarcate and sort CD31hiEMCNhi cells, first standard quadrant gates were set. To differentiate CD31hiEMCNhi cells from the total double positive cells in quadrant, two gates were set at >104 log Fl-2 (PE-Cy7-CD31) fluorescence and >104 log Fl-4 (Alexa Fluor 647-Endomucin) fluorescence. DAPI−CD45−Ter119−CD31+ cells were sorted according to side scatter and were set at >103 log Fl-2 fluorescence (PE-Cy7-CD31) after negative selection of CD45 and Ter119 at <103 log Fl-2 (PE-CD45 and APC-Ter119) fluorescence. DAPI−CD45−Ter119−CD31+ cells were sorted as total bone ECs.
Ter119 apc
Manufactured by BD
Ter119-APC is a fluorescently labeled antibody that binds to the Ter119 antigen, which is expressed on red blood cells and their precursors. It is used as a cell surface marker in flow cytometry applications to identify and analyze erythroid cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd31 apc, by BD (7 mentions)
Facscalibur, by BD (5 mentions)
Cd45 apc, by BD (4 mentions)
Cd71 fitc, by BD (4 mentions)
Sytox blue, by Thermo Fisher Scientific (3 mentions)
Cd24 pe, by BD (3 mentions)
Anti epcam pe cy7, by BioLegend (2 mentions)
Anti cd49f fitc, by BioLegend (2 mentions)
Facsaria, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Cd71 pe, by BD (2 mentions)
C kit apc, by BD (2 mentions)
Cd45 apc, by BioLegend (2 mentions)
Cd29 fitc, by BD (2 mentions)
Facsaria 2 cell sorter, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Cd45 pe, by BD (1 mentions)
Endomucin antibody, by Santa Cruz Biotechnology (1 mentions)
19 protocols using ter119 apc
1
Isolation of Bone-Derived Endothelial Cells
2
Mammary Gland Cell Isolation and Flow Cytometry
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Flow cytometry was performed as described16 (link),17 (link). Briefly, mammary glands were isolated, minced with a razor blade, and incubated with 300 U/mL collagenase (Sigma, C9891) and 100 U/mL hyaluronidase (Sigma, H3506) for 90 min at 37 °C. The cells were treated with red blood cell lysis buffer (Sigma, R7757) for 5 min at room temperature before treating with 0.25% Trypsin (Gibco, 25,200) for 2 min at 37 °C and dispase II (2 mg/mL) (Stem Cell Technologies, 07,913) with 0.1 mg/mL DNase I (Sigma, DN25) for 2 min at 37 °C. Cells were stained for 30 min in the dark at room temperature using APC-CD45 (BD Biosciences, 559,864; 1:250), APC-CD31 (BD Biosciences, 551,262; 1:250), APC-Ter119 (BD Biosciences, 557,909; 1:250), PE/C7-Epcam (Bio Legend, 118,215; 1:250), and PerCP-Cy5.5-Cd49f (Bio Legend, 555,735; 1:250). Cells were washed and stained with Sytox blue (Invitrogen, S34857; 1:1000) before flow cytometry.
3
Multicolor Flow Cytometry of B Cells
4
Single Cell Phenotyping of Mammary Epithelium
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Single cell suspension from above was stained using the following antibodies and reagents: anti-CD49f-FITC (1:250, Bio Legend, 102205), anti-EpCAM-PE-Cy7 (1:250, Bio Legend, 118215), anti-lineage-APC [1:250; including APC-CD45 (BD Biosciences, 559864), APC-CD31 (BD Biosciences, 551262), APC-TER119 (BD Biosciences, 557909)], and SytoxBlue (Invitrogen, S3457). Flow cytometry analysis and sorting were performed on a Novocyte and FACSAria (Becton Dickenson UK), respectively.
5
Multiparametric Flow Cytometry Analysis of Organ Cells
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Flow cytometry was used to quantify the percentage of cells in organs that were Cy7 positive or td-Tomato positive following treatment. Single-cell suspensions were obtained as described previously [49 ,50 ] for further staining and flow cytometry analysis. Freshly dissected the heart, the liver, lungs, kidneys, the GI tract, and the brain were minced and digested with 500 μL of 1 mg/mL collagenase type I (Gibco) by incubating at 37 °C, 5% CO2 for 20 min in 1.5 ml Eppendorf tubes. Cell suspension was collected, neutralized with a medium containing 10% fetal bovine serum (FBS), and placed on ice. Fresh collagenase solution was added to the remaining tissue and incubated for an additional 20 min. Cell suspensions were filtered through 70um Nylon cell strainer (Falcon, cat. 352,350) to create a single-cell suspension. The Attune NxT Flow Cytometer (Thermo Fisher Scientific) was used for performing flow cytometry, and FlowJo software (FlowJo LLC) was used for data analyses. All antibodies were obtained from BD Biosciences. Cells were stained with BV421-CD56 (cat.748,094), APC-CD31 (551,262), FITC-CD90.2 (553,003), BV421-CD45 (563,890), BV650 CD324 (752,472), FITC-CD71 (561,936), APC-Ter119 (557,909) from BD Biosciences. BDTM anti-Rat Ig, κ CompBeads (552,844) were used to generate compensation controls.
6
Single Cell Staining for Flow Cytometry
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Single cell suspension from above was stained using the following antibodies and reagents: anti-CD49f-FITC (1:250, Bio Legend, 102205), anti-EpCAM-PE-Cy7 (1:250, Bio Legend, 118215), anti-lineage-APC [1:250; including APC-CD45 (BD Biosciences, 559864), APC-CD31 (BD Biosciences, 551262), APC-TER119 (BD Biosciences, 557909)], and SytoxBlue (Invitrogen, S3457). Flow cytometry analysis and sorting were performed on a FACSAria (Becton Dickenson UK).
7
Comprehensive Multiparameter Flow Cytometry Protocol
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Single‐cell suspensions from the spleen and bone marrow were counted using counting slides (SD‐100, Nexcelom) in a Cellometer Mini Automated Cell Counter (Nexcelom). Surface antigens were stained with demonstrated conjugated primary antibodies in the staining buffer (1× PBS, 3% BSA, 1 mM EDTA, 0.1% NaN3) at 4 °C for 30 min. Antibodies used were as follows: APC TER‐119 (557 909, BD), PE CD71 (553 267,BD), Biotin CD3 (100 244, Biolegend), Biotin CD45R (13‐0452‐81, eBioscience),Biotin CD4 (13‐0041‐81, eBioscience), Biotin CD5 (13‐0051‐81, eBioscience), Biotin CD8a Biotin (13‐0081‐81, eBioscience),Biotin CD19 (13‐0193‐81, eBioscience),Biotin CD11b (13‐0112‐81, eBioscience),Biotin Ly‐6G (13‐5931‐81, eBioscience), Biotin TER‐119 (13‐5921‐81, eBioscience), PE‐Cy7 Streptavidin (25‐4317‐82, eBioscience), APC Ly‐6A/E (Sca‐1) (17‐5981‐82, eBioscience), APC‐EFLUOR CD117 (C‐KIT) (47‐1172‐82, eBioscience). After staining, cells were washed once with 1× PBS and quickly analyzed by a cytoflex S flow cytometer (cytoflex S, Beckman Coulter). All analyses were performed using CytExpert software (CytExpert, Beckman Coulter, Inc.).
An auto hematology analyzer (BC‐2800Vet, Mindray) was used to analyze diluted peripheral blood that had been collected in an anticoagulation tube and diluted in EDTA buffer (0.5 M EDTA, pH 8.0) at a quantitative ratio of 1:1.
An auto hematology analyzer (BC‐2800Vet, Mindray) was used to analyze diluted peripheral blood that had been collected in an anticoagulation tube and diluted in EDTA buffer (0.5 M EDTA, pH 8.0) at a quantitative ratio of 1:1.
8
Isolation and Analysis of Fetal Liver Cells
9
Multiparametric Flow Cytometry Analysis of Hematopoietic Cells
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BM cells or splenocytes were harvested and subjected to red blood cell lysis. Fresh or frozen cells were stained with the following Abs: CD45.2-FITC and CD45.1-APC, Mac1-PE, Gr1-APC, c-Kit–APC, CD71-PE, Ter119-APC, B220-PE, and CD3-APC (BD) and analyzed on the BD FACSCalibur instrument. Staining for multiparameter flow cytometry was performed after a c-kit enrichment using 10 µl MACS beads (CD117) per mouse and then run on an AutoMACS (Miltenyi Biotec) according to the manufacturer’s instructions. The cells were then stained with the following cocktail: (Lineage; CD3, CD4, CD8, Gr1, B220, CD19, and TER119 all conjugated with PeCy5), Sca-Pac Blue, CD34-FITC or CD45.2-FITC, SLAM-APC, CD48-PE, c-KIT–Alexa Fluor 780, and FcgRIIb-PeCy7 (Fig. 2 d ); HPCs (Linloc-Kit+Sca1−), GMPs (LK, FcγRIIbhiCD34+), CMPs (LK, FcγRIIbmid CD34+), MEPs (LK, FcγRIIbloCD34−), B cells (B220+), and T cells (CD3+) from the spleen were also sorted. For analysis of LMPPs and CLPs, the following cocktail was used: Lineage marker mix–PeCy5, Sca-Pecy7 IL-7Ra–Pac Blue, Flk2-PE, CD34-FITC, and Kit-APC (Martin et al., 2003 (link); Adolfsson et al., 2005 (link); Karsunky et al., 2008 (link)).
10
Breast Cancer Stem Cell Characterization
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Cells were stained with Aldefluor reagent as per manufacturer's instructions (Stemcell Technologies, Cat# 01700) and the percentage of cells with high Aldehyde Dehydrogenase (ALDH) activity were analyzed. Human breast cancer stem cell marker staining was determined by labeling human cells with CD44-APC and CD24-PE antibodies (BD Biosciences, Cat# 559942 and 555428). Dissociated mammary tumor cells and murine cell lines were stained with CD24-PE, CD29-FITC, CD31-APC, TER-119-APC (BD Biosciences, Cat# 553262, 561796, 561814, and 561033) and CD45-APC (Biolegend, Cat# 103111) antibodies, analyzed using the FACS Diva software, and sorted using the Aria Illu sorter (BD Biosciences) [47 (link)].
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