Ter119 pe cy5

Manufactured by Thermo Fisher Scientific

The Ter119-PE/Cy5 is a laboratory reagent that can be used for the detection and analysis of Ter119-positive cells in flow cytometry applications. It is a fluorescently-labeled antibody that binds to the Ter119 antigen, which is expressed on erythroid cells. The Ter119-PE/Cy5 reagent allows for the identification and enumeration of Ter119-positive cells in a sample.

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Ter119 (107 citations) Ter119-PE (12 citations)

6 protocols using ter119 pe cy5

Isolation and identification of myeloid cells

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Myeloid cells were extracted from whole hemispheres, isolated into single-cell suspensions and identified using fluorescence-activated cell sorting (FACS) gating for CD11b⁺CD45^int as previously described (Elmore et al., 2014 (link)). Cells were stained with the following surface antibodies purchased from Biolegend (San Deigo, CA) at 1:200 unless otherwise indicated: CD34-eFlour660 (1:50, 50-0341-80, eBioscience, San Diego, CA), Sca-1-AF700 (1:100, 108141), CD16/32-PE (101307), Ter119-PE/Cy5 (116209), ckit/CD117-PE/Cy7 (25-1171-81, eBioscience), CD150/SLAM-PerCP-eFlour710 (46-1502-82, eBioscience), CD11b-APC (101212), CD11b-PE (101208), Gr1-AF700 (108422), CD45-AF700 (103128), CD45-APC/Cy7 (103116), NK1.1-PE (108707), CD3-PE/Cy7 (100220), CD19-Per-Cyanine5.5 (45-0193-82, eBioscience), CD11c-APC/Cy7 (117323), Ly6C-PE (1:400, 128007), Ly6G-5.5 (127615). For HSCs, CMPs, and GMPs, all cells were gated on live (PI^-), Ter119^- cells and then identified with the following gating strategy: HSCs: FcyR^-, ckit⁺ Sca⁺ CD34^-, SLAM⁺, CMPs: FcyR^-, ckit⁺, Sca^-, CD35⁺, and GMPs: FcyR⁺, ckit⁺, Sca^-, CD34⁺. Samples were acquired with the BD LSRII or BD Fortessa X20, and sorted with the BD FACS Aria II.

Hohsfield L.A., Najafi A.R., Ghorbanian Y., Soni N., Crapser J., Figueroa Velez D.X., Jiang S., Royer S.E., Kim S.J., Henningfield C.M., Anderson A., Gandhi S.P., Mortazavi A., Inlay M.A, & Green K.N. (2021). Subventricular zone/white matter microglia reconstitute the empty adult microglial niche in a dynamic wave. eLife, 10, e66738.

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Hematopoietic Stem Cell Transplant Protocol

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B6.SJL-Ptprca mice were used at 6–10 wk of age. Mice were irradiated with a split dose of 9GY before transplantation. Each recipient received 1 embryo equivalent of sorted cells via tail vein injection, along with 2 × 10⁴ bone marrow or 2 × 10⁵ splenic helper cells per experiment group from B6.SJL-Ptprca mice unless indicated otherwise. For secondary transplantations, CD45.2⁺ cells were isolated from recipient bone marrow and 3 × 10⁵ cells were transplanted per recipient with 2 × 10⁵ splenic helper cells. Peripheral blood was collected retroorbitally at the indicated time point after transplantation. Red blood cells were removed with 1% dextran sulfate/0.5% EDTA/PBS (wt/vol) and treated with RBC lysis buffer (Sigma-Aldrich). Leukocytes were then stained in 2% serum/PBS (vol/vol) for CD45.1-FITC (A20; BD), CD45.2-PE-Cy7 (104; BioLegend), Mac1-Alexa Fluor 700 (M1/70; BioLegend), Gr1-PE (A20; BD), CD19-APC-Cy7 (6D5; BioLegend), CD3-APC (145-2C11; eBioscience), Ter119-PE-Cy5 (TER-119; eBioscience), B220-Pacific Blue (RA3-6B2; BD), and propidium iodide (PI). Engraftment was determined to be a percentage of PI and Ter119-negative CD45.2 cells within the CD45-positive population.

Kim P.G., Nakano H., Das P.P., Chen M.J., Rowe R.G., Chou S.S., Ross S.J., Sakamoto K.M., Zon L.I., Schlaeger T.M., Orkin S.H., Nakano A, & Daley G.Q. (2015). Flow-induced protein kinase A–CREB pathway acts via BMP signaling to promote HSC emergence. The Journal of Experimental Medicine, 212(5), 633-648.

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Fetal Hematopoietic Stem Cell Transplantation

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Fetal HSCs (Lin-/Sca-1+/c-Kit+/Flk2-/CD150+/CD48-) were isolated from the fetal livers of E14.5 Mcm3^+/+, Mcm3^+/Lox or Mcm3^Lox/Lox CD45.2 C57Bl/6 donor mice, and transplanted into lethally irradiated CD45.1 C57Bl/6 primary recipients (50 fetal HSCs per mouse; 5 mice per group) together with 300,000 Sca-1-depleted CD45.1 helper BM cells. 4 months post-transplantation, the primary recipients were sacrificed to assess the percentage of donor-derived chimerism in the bone marrow (BM), spleen, peripheral blood and HSC compartment and to re-isolate donor-derived adult MCM3^+/+ or MCM3^Lox/Lox HSCs53 (link). Donor chimerism was analyzed using CD45.2-FITC, (eBioscience 12-0453-83; 1:50), B220-APC-e780 (47-0452-82; 1:800), Gr-1-PB (1:400), Mac-1-PE-Cy7 (1:3200), CD3-e660 (eBioscience, 50-0032-82; 1:400) and Ter-119-PE-Cy5 (eBioscience 15-5921-83; 1:800) antibodies. Re-isolated CD45.2⁺ donor-derived adult MCM3^+/+ or MCM3^Lox/Lox HSCs were transplanted into lethally irradiated CD45.1 secondary recipients (500 HSCs per mouse; 5 mice per group) together with 300,000 Sca-1-depleted CD45.1 helper BM cells.

Alvarez S., Díaz M., Flach J., Rodriguez-Acebes S., López-Contreras A.J., Martínez D., Cañamero M., Fernández-Capetillo O., Isern J., Passegué E, & Méndez J. (2015). Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality. Nature Communications, 6, 8548.

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Characterizing Fetal HSC Function via Transplantation

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Fetal HSCs (Lin−/Sca-1+/c-Kit+/Flk2−/CD150+/CD48−) were isolated from the fetal livers of E14.5 Mcm3^+/+, Mcm3^+/Lox or Mcm3^Lox/Lox CD45.2 C57Bl/6 donor mice, and transplanted into lethally irradiated CD45.1 C57Bl/6 primary recipients (50 fetal HSCs per mouse; 5 mice per group) together with 300,000 Sca-1-depleted CD45.1 helper BM cells. 4 months post-transplantation, the primary recipients were sacrificed to assess the percentage of donor-derived chimerism in the bone marrow (BM), spleen, peripheral blood and HSC compartment and to re-isolate donor-derived adult MCM3^+/+ or MCM3^Lox/Lox HSCs^{53 (link)}. Donor chimerism was analyzed using CD45.2-FITC, (eBioscience 12-0453-83; 1:50), B220-APC-e780 (47-0452-82; 1:800), Gr-1-PB (1:400), Mac-1-PE-Cy7 (1:3200), CD3-e660 (eBioscience, 50-0032-82; 1:400) and Ter-119-PE-Cy5 (eBioscience 15-5921-83; 1:800) antibodies. Re-isolated CD45.2⁺ donor-derived adult MCM3^+/+ or MCM3^Lox/Lox HSCs were transplanted into lethally irradiated CD45.1 secondary recipients (500 HSCs per mouse; 5 mice per group) together with 300,000 Sca-1-depleted CD45.1 helper BM cells.

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Phenotypic Characterization of Murine Hematopoietic Stem Cells

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Bone marrow/hematopoietic stem cells were extracted from femurs and tibia by flushing with PBS + 2% FBS. Peripheral blood cells/granulocytes were collected via the tail vein in EDTA. Red blood cells were lysed using 1x ACK Lysis Buffer. Cells were then stained for analysis by flow cytometry with the following surface antibodies purchased from Biolegend at 1:200 unless otherwise indicated: CD34-eFlour660 (1:50, 50-0341-80, eBioscience), Sca-1-AF700 (1:100, 108141), Ter119-PE/Cy5 (116209), ckit/CD117-PE/Cy7 (25-1171-81, eBioscience), CD150/SLAM-PerCP-eFlour710 (46-1502-82, eBioscience), CD11b-APC (101212), Gr1-AF700 (108422), CD45-APC/Cy7 (103116), NK1.1-PE (108707), and CD27-APC/Cy7 (124226). Flow cytometry analysis was performed using the BD LSRII.

Hohsfield L.A., Najafi A.R., Ghorbanian Y., Soni N., Hingco E.E., Kim S.J., Jue A.D., Swarup V., Inlay M.A, & Green K.N. (2020). Effects of long-term and brain-wide colonization of peripheral bone marrow-derived myeloid cells in the CNS. Journal of Neuroinflammation, 17, 279.

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Murine Hematopoietic Stem Cell Characterization

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Bone marrow/Hematopoietic stem cells were extracted from femurs and tibia by ushing with PBS + 2% FBS. Peripheral blood cells/granulocytes were collected via the tail vein in EDTA. Red blood cells were lysed using 1x ACK Lysis Buffer. Cells were then stained for analysis by ow cytometry with the following surface antibodies purchased from Biolegend at 1:200 unless otherwise indicated: CD34-eFlour660 (1:50, 50-0341-80, eBioscience), Sca-1-AF700 (1:100, 108141),, Ter119-PE/Cy5 (116209), ckit/CD117-PE/Cy7 (25-1171-81, eBioscience), CD150/SLAM-PerCP-eFlour710 (46-1502-82, eBioscience), CD11b-APC (101212), Gr1-AF700 (108422), CD45-APC/Cy7 (103116), NK1.1-PE (108707) and CD27-APC/Cy7 (124226). Flow cytometry analysis was performed using the BD LSRII.

Hohsfield L.A., Najafi A.R., Ghorbanian Y., Soni N., Hingco E., Kim S.J., Jue A.D., Swarup V., Inlay M.A., & Green K.N. (2020). Effects of long-term and brain-wide colonization of peripheral bone-marrow derived myeloid cells in the CNS.

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