Apc anti mouse ter119

Manufactured by BioLegend

Sourced in United States

APC anti-mouse Ter119 is a monoclonal antibody that binds to the Ter119 antigen, which is expressed on mouse erythroid cells. This antibody is conjugated with the fluorescent dye Allophycocyanin (APC).

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9 protocols using apc anti mouse ter119

Antibody Staining for Flow Cytometry

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Anti-TRIM33 antibody (A301-060A; Bethyl Laboratories, Montgomery, TX), Anti-PU.1 antibody (#2266; Cell Signaling, Beverly, MA or sc-352; Santa Cruz, Dallas, TX), Anti-Bim antibody (#2819; Cell Signaling), Anti-H3K27ac antibody (ab4729; Abcam, Cambridge, MA), Anti-H3K4me3 antibody (07-473; Millipore), Anti-ß-actin HRP antibody (#A3854; Sigma, Ronkonkoma, NY), APC anti-mouse B220 (#103212; BioLegend, San Diego, CA), APC anti-mouse CD-19, APC anti-mouse Mac-1/Cd11b (#101211; BioLegend), APC anti-mouse Ly-6G/Gr-1 (#17-5931; eBioscience, San Diego, CA), APC anti-mouse TER-119 (#116212; BioLegend), APC anti-mouse CD-3 (#100209; BioLegend).

Wang E., Kawaoka S., Roe J.S., Shi J., Hohmann A.F., Xu Y., Bhagwat A.S., Suzuki Y., Kinney J.B, & Vakoc C.R. (2015). The transcriptional cofactor TRIM33 prevents apoptosis in B lymphoblastic leukemia by deactivating a single enhancer. eLife, 4, e06377.

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Leukocyte and Erythrocyte Flow Cytometry

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Mouse bone marrow cells were prepared as described above. Splenocytes were isolated as previously described^{51 (link)}. For flow cytometry analysis of the leukocytes, red blood cells were depleted with red blood cell lysis buffer (Roche). Cells (0.5–1 × 10⁶) were incubated with Fc block (anti-mouse CD16/32, BioLegend) to block nonspecific Fc binding, stained with isotype control or FITC-anti-mouse CD11b (eBioscience) and PE-anti-mouse Ly-6G/Ly-6C (Gr1) antibody (Biolegend) according to the supplier’s instructions. For flow cytometry analysis of red blood cells, whole mouse bone marrow cells or splenocytes were stained with isotype control or APC anti-mouse Ter119 (BioLegend) and BV-421 anti-mouse CD71 antibody (BD), according to the supplier’s instructions. Flow cytometry was performed using BD LSRFortessa or BD FACSCanto II Flow Cytometer system, followed by analysis with FlowJo software (Tree Star, Inc.).

Wang C., Xu C.X., Alippe Y., Qu C., Xiao J., Schipani E., Civitelli R., Abu-Amer Y, & Mbalaviele G. (2017). Chronic inflammation triggered by the NLRP3 inflammasome in myeloid cells promotes growth plate dysplasia by mesenchymal cells. Scientific Reports, 7, 4880.

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Multimarker Identification of Stem Cells

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Single cells were labeled with the required antibodies for 20 min at 4°C, washed, and re-suspended in DMEM/F12 medium containing 10 Mm HEPES, 5% FBS, and 1 mg/mL DAPI before analysis. The antibodies used were: biotin anti-CD133 (1:100; BioLegend), PE/Cy7 anti-mouse CD24 (1:100, BioLegend), PerCP/Cy5.5 anti-mouse Sca-1 (1:100, BioLegend), AlexaFluor700 anti-mouse/rat CD29 (1:100, BioLegend), PE anti-mouse CD49b (1:50, BioLegend); lineage markers: APC anti-mouse CD31 (1:100, BioLegend), APC anti-mouse Ter119 (1:100, BioLegend), APC anti-mouse CD45 (1:100, BioLegend); APC/Cy7 Streptavidin; isotype controls: PE rat IgM (1:50; BioLegend), PerCP/Cy5.5 rat IgGa (1:100, BioLegend). Single live cells were gated by DAPI exclusion and analyzed or sorted on LSRII, FACS Vantage or FACS Aria flow cytometers. The purity of sorted populations was about 95%.

Rodilla V., Dasti A., Huyghe M., Lafkas D., Laurent C., Reyal F, & Fre S. (2015). Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development. PLoS Biology, 13(2), e1002069.

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Multiparametric Analysis of Erythroid Lineage

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Spleen, BM, enriched EBIs, or EDTA treated blood samples were isolated, single cell suspensions generated by filtering through 70 μm mesh, and 1 × 10⁶ cells were stained with Zombie Aqua^™ Fixable Viability dye (BioLegend, CA) for 15 minutes at room temperature, washed twice with FACS buffer (PBS +2%FBS) and fixed with 1% PFA for 1 hour on ice. Cells were washed with FACS buffer and pellets resuspended in PE anti-mouse CD71 (BioLegend, CA, 1:200), APC anti-mouse Ter119 (BioLegend, CA, 1:200) and FITC anti-mouse F4/80 (BioLegend, CA, 1:200) for 30 minutes on ice protected from light. Cells were washed twice in FACS buffer, resuspended in ethanol at −20 °C for 1 hour and stained with Hoechst 33342 Solution (Thermo Fisher Scientific, MA, 1:1000) for 15 minutes before flow cytometry analysis was performed using a BD LSRFortessa^™ X-20 (BD Biosciences, CA). Results were analyzed using FlowJo® v10.4.2 (FlowJo, LLC, CA).

Rego S.L., Harvey S., Simpson S.R., Hemphill W.O., McIver Z.A., Grayson J.M, & Perrino F.W. (2018). TREX1 D18N mice fail to process erythroblast DNA resulting in inflammation and dysfunctional erythropoiesis. Autoimmunity, 51(7), 333-344.

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Multiparameter Flow Cytometry for Murine B Cell Analysis

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B cell developmental analyses were carried out in 8–12-weeks-old mice. Single-cell suspensions were prepared from the bone marrow (BM), and spleen (Spl) of the mice of the indicated genotypes, and ~1 × 10⁵ cells in 1X PBS were stained for 15 min at RT using fluorescence-conjugated antibodies. Cells were analyzed by flow cytometry on a FACSCalibur flow cytometer (BD Biosciences), and data were processed using the FlowJo software package. The following antibody cocktail was used for B cell analyses, with each antibody used at 1:200 dilution: FITC anti-mouse CD43 (Biolegend, 553270); PE anti-mouse IgM (Southern Biotech, 1020-09); PE-Cyanine5 anti-human/mouse CD45R (B220), (eBioScience, 15-0452-83); and APC anti-mouse TER119 (Biolegend, 116212). Live cells were gated based on forward, and side scatters (FSC and SSC, respectively). Red blood cells (RBC) were excluded using a Ter119 antibody specific for RBC.

Menolfi D., Lee B.J., Zhang H., Jiang W., Bowen N.E., Wang Y., Zhao J., Holmes A., Gershik S., Rabadan R., Kim B, & Zha S. (2023). ATR kinase supports normal proliferation in the early S phase by preventing replication resource exhaustion. Nature Communications, 14, 3618.

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Multiparametric Flow Cytometry Analysis of Immune Cell Development

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Mouse blood cell and lymphocyte development were analyzed by flow cytometry (36 (link), 37 (link)). B lymphocyte development was conducted by staining with cocktails including FITC anti-mouse CD43 (Biolegend, 553270), PE goat anti-mouse IgM (Southern Biotech, 1020-09), PE-cyanine5 anti-Hu/Mo CD45R (B220) (eBioScience, 15-0452-83), and APC anti-mouse TER119 (Biolegend, 116212). T lymphocyte development was measured on thymocytes using cocktail including PE rat anti-mouse CD4 (BD Pharmingen, 557308), FITC anti-mouse CD8a (Biolegend, 100706), PE/Cy5 anti-mouse CD3e (eBioscience, 15-0031-83), and APC anti-mouse TCRβ (BD Pharmingen, 553174). Myeloid cells were measured in bone marrow cells and splenocytes using cocktail including FITC anti-mouse CD11b (BD Pharmingen, 553310), PE rat anti-mouse CD19 (BD Pharmingen, 557399), PE/Cy5 anti-mouse CD3e (eBioscience, 15-0031-83), and APC anti-mouse Ly6G/Ly6C(Gr-1) (Biolegend, 108412). Purified splenocyte CSR was stained with PE-cyanine5 anti-Hu/Mo CD45R (B220) and FITC rat anti-mouse IgG1(BD Pharmingen, 553443). All antibodies were diluted according to the manufacturer’s protocol. The flow cytometry data were collected on either an LSR II (BD), or on an Attune NxT (Invitrogen) flow cytometer. All flow cytometry data were analyzed using FlowJo V10.

Shao Z., Lee B.J., Zhang H., Lin X., Li C., Jiang W., Chirathivat N., Gershik S., Shen M.M., Baer R, & Zha S. (2023). Inactive PARP1 causes embryonic lethality and genome instability in a dominant-negative manner. Proceedings of the National Academy of Sciences of the United States of America, 120(31), e2301972120.

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Isolation and Characterization of Murine Hematopoietic Cells

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Spleen, femur, and tibia were isolated from the mice. Spleen samples were ground and filtered through a 70 μm filter. Bone marrow samples were obtained by destroying femurs and tibias. All mice samples were incubated in PBS with 2% FBS to keep alive. After centrifugation, the suspended cells in PBS were stained with Fixable Viability Stain 510 live/dead dye (BD Bioscience, San Diego, CA, United States) and mixed cell surface antibodies, respectively, including APC-Cy™7-Rat anti-mouse CD45 (BD Bioscience, San Diego, CA, United States), PE anti-mouse CD71 (Biolegend, San Diego, CA, United States), APC anti-mouse Ter119 (Biolegend), FITC anti-mouse Lineage Cocktail (Biolegend), PE-Cy7 anti-mouse Sca-1 (Biolegend), and Brillant Violet 421 anti-mouse CD117 (Biolegend). Finally, all samples were analyzed by FACS Aria machine (BD Bioscience). Fluorescence-minus-one (FMO) controls were included for all the flow cytometry analyses.

Bai M., Cao P., Lin Y., Yu P., Song S., Chen L., Wang L, & Chen Y. (2022). Intermittent Caloric Restriction Promotes Erythroid Development and Ameliorates Phenylhydrazine-Induced Anemia in Mice. Frontiers in Nutrition, 9, 892435.

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Single-cell isolation and flow cytometry

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Single cell dissociation was performed through enzymatic digestion with 600 U/mL collagenase (Sigma) and 200 U/mL hyaluronidase (Sigma) for 1 h at 37°C. Cells were further dissociated in TrypLE (Gibco) for 3 min, in 5 mg/mL dispase (Roche) and 0.1 mg/mL DNase I (Sigma) for 5 min, and then in 0.63% NH4Cl and filtered through a 40 μm cell strainer to obtain a single cell preparation for FACS. Cell labelling, and flow cytometry were performed as previously described in Rodilla et al., 2015. Dead cells (DAPI+), and CD45+/CD31+/Ter119+ (Lin+) non-epithelial cells were excluded before analysis using LSRII or FACS ARIA flow cytometers (BD). The following antibodies were used in 1:100 final concentration: biotin anti-CD133 (BioLegend), PE/Cy7 anti-mouse CD24 (BioLegend), PerCP/Cy5.5 anti-mouse Sca-1 (BioLegend), AlexaFluor700 anti-mouse/rat CD29 (BioLegend), PE anti-mouse CD49b (BioLegend); lineage markers: APC anti-mouse CD31 (BioLegend), APC anti-mouse Ter119 (BioLegend), APC anti-mouse CD45 (BioLegend); APC/Cy7 Streptavidin; isotype controls: PE rat IgM (BioLegend), PerCP/Cy5.5 rat IgGa (BioLegend). The purity of sorted populations was about 95%. The results were analyzed using FlowJo software and the data processing with Prism-graphpad.

Lilja A.M., Rodilla V., Huyghe M., Hannezo E., Landragin C., Renaud O., Leroy O., Rulands S., Simons B.D, & Fre S. (2018). Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland. Nature cell biology, 20(6), 677-687.

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Bone Marrow Leukocyte Immunophenotyping

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Mouse bone marrow cells were flushed from femur and tibia. For flow cytometry analysis of the leukocytes, red blood cells (RBCs) were depleted with RBC lysis buffer (Roche, Brighton, MA). Cells (0.5–1 × 10⁶) were incubated with Fc block (anti-mouse CD16/32, BioLegend, San Diego, CA) to block nonspecific Fc binding, stained with isotype control or APC-anti-mouse Ter119 (BioLegend, San Diego, CA), FITC-anti-mouse CD11b (eBioscience, Grand Island, NY), and PE-anti-mouse Ly-6G/Ly-6C (Gr1) antibody (BioLegend, San Diego, CA) according to the supplier’s instructions. Flow cytometry was performed using BD LSRFortessa or BD FACSCanto II Flow Cytometer system, followed by analysis with FlowJo software (Tree Star, Ashland, Oregon).

Xiao J., Wang C., Yao J.C., Alippe Y., Xu C., Kress D., Civitelli R., Abu-Amer Y., Kanneganti T.D., Link D.C, & Mbalaviele G. (2018). Gasdermin D mediates the pathogenesis of neonatal-onset multisystem inflammatory disease in mice. PLoS Biology, 16(11), e3000047.

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