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Sca 1 clone d7

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Sca-1 (clone D7) is a monoclonal antibody that recognizes the Sca-1 (Stem cell antigen-1) marker. Sca-1 is a widely used marker for the identification and isolation of murine hematopoietic stem and progenitor cells.

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Lab products found in correlation

Cd11b, by BioLegend (3 mentions) Lsr 2, by BD (3 mentions) Sh800, by Sony (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Cd172α, by BioLegend (2 mentions) Gr1 clone rb, by BioLegend (2 mentions) Ly6c clone hk, by BioLegend (2 mentions) Siglec h clone ebio440c, by BioLegend (2 mentions) Ly6g clone 1a8, by BioLegend (2 mentions) Ter119, by BioLegend (1 mentions) C kit clone 2b8, by Thermo Fisher Scientific (1 mentions) Ly6g 6c, by BioLegend (1 mentions) Facsaria 2, by BD (1 mentions) Streptavidin efluor 450, by Thermo Fisher Scientific (1 mentions) Epcam clone g8, by BioLegend (1 mentions) Cd31 biotin clone 390, by Thermo Fisher Scientific (1 mentions) Cd45 biotin clone 30 f11, by Thermo Fisher Scientific (1 mentions) Ter119 biotin clone ter119, by Thermo Fisher Scientific (1 mentions) Cd49b hmα2, by BioLegend (1 mentions) Facsaria 2 flow cytometer, by BD (1 mentions)

Spelling variants of this product

Sca-1 (56 citations) Clone D7 (4 citations) Anti–Sca-1 (clone D7; (4 citations) Sca-1-Pacific Blue (clone D7; (2 citations)

7 protocols using sca 1 clone d7

1

Murine Hematopoietic Stem Cell Characterization

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Total bone marrow (BM) from one femur and two tibiae was isolated by flushing. After cell counting 1 × 106 cells were stained with a cocktail for lineage markers (CD3, CD11b, B220, Ter119, and Ly6G/6C; BioLegend), and lineage-negative cells (Lin-) were analyzed for c-Kit (clone 2B8; eBioscience) and Sca-1 (clone D7; BioLegend) expression to discriminate Kit+Sca-1- (KL) and Kit+Sca-1+ (KSL) cells. Progenitor and stem cell subpopulations were identified by staining with the following additional markers: CD34 (clone MEC14.7; BioLegend), Fc-gamma-II/III-R (clone 93; eBioscience).
Klein-Rodewald T., Micklich K., Sanz-Moreno A., Tost M., Calzada-Wack J., Adler T., Klaften M., Sabrautzki S., Aigner B., Kraiger M., Gailus-Durner V., Fuchs H., Gründer A., Pahl H., Wolf E., Hrabe de Angelis M, & Rathkolb B. (2022). New C3H KitN824K/WT cancer mouse model develops late-onset malignant mammary tumors with high penetrance. Scientific Reports, 12, 19793.
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2

Mammary Cell Immunophenotyping Protocol

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Single mammary cells were then incubated with the following primary antibodies: CD31-biotin (clone 390, eBioscience), CD45-biotin (clone 30-F11, eBioscience), Ter119-biotin (clone Ter119, eBioscience), EpCAM (clone G8.8, BioLegend), CD49f (clone GoH3, BioLegend), CD49b (HMα2, BioLegend), and Sca1 (clone D7, BioLegend). Biotin conjugated antibodies were detected with Streptavidin-eFluor450 (eBioscience). Cells were then filtered through a 30-μm cell strainer (Partec) and incubated with 4’, 6-diamidino-2-phenylindole (DAPI; Invitrogen) and were analysed by using an LSRII (Becton Dickinson), or sorted on a FACSAria II (Becton Dickinson). The gating strategy to select luminal and basal subsets is shown in Supplementary Fig. 5a. Flow cytometry data were analysed using FlowJo (version 10. Tree Star Inc.)
Shehata M., Waterhouse P.D., Casey A.E., Fang H., Hazelwood L, & Khokha R. (2018). Proliferative heterogeneity of murine epithelial cells in the adult mammary gland. Communications Biology, 1, 111.
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3

Isolation and Characterization of Islet Cells

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Cultured MSCs were detached by incubating with 0.05% trypsin/EDTA solution at 37°C for 5 minutes. Single cells suspension was obtained by pass the detached MSCs through a 0.45μm cell strainer. For FACS analysis of islet cells, fresh isolated islets were treated with 0.05% Trypsin/EDTA for 5 min at 37°C, followed by pipetting to separate single cells. The cells are then incubated with purified anti-FcγR (clone 2.4G2, BD Biosciences) before staining with fluorescent antibodies, including CD45 (clone 30-F11, BD Biosciences), CD31 (clone MEC13.3, BioLegend), and CD105 (clone MJ7/18, BioLegend) and Sca-1 (clone D7, BioLegend). Samples were analyzed on a BD LSR II or a FACScalibur (BD Biosciences), and cell sorting was performed on a FACS Aria II flow cytometer.
Dai Y.D., Dias P., Margosiak A., Marquardt K., Bashratyan R., Hu W.Y., Haskins K, & Evans L.H. (2020). Endogenous Retrovirus Gag Antigen and Its Gene Variants Are Unique Autoantigens Expressed in the Pancreatic Islets of Non-obese Diabetic Mice. Immunology letters, 223, 62-70.
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4

Murine Acute Promyelocytic Leukemia Model

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An in vivo model of murine APL was prepared as previously described [139 (link)]. Briefly, leukemic spleen cells from hMRP8 PML-RAR transgenic mice were transplanted by retro-orbital injection into sub-lethally irradiated 12 week old FVB/N mice. Spleen cells from either leukemic or wild-type control mice were sorted using an Influx cell sorter based on expression of the following cell surface markers: Sca1 (clone D7; Biolegend), CD45/B220 (RA3-6B2; Biolegend), CD19 (MB19-1; Biolegend), CD3 (145-2C11, Biolegend) antigens, all pacific blue conjugated (omitting Gr1 antibodies from the usual depletion cocktail). The depletion cocktail-negative cells were then separated using antibodies against c-kit conjugated with allophycocyanin (clone 2B8; Biolegend) and CD34 conjugated with fluorescein isothiocyanate (RAM34; eBioscience). LICs from leukemic mice were characterized by depletion cocktail-, c-kit+, and CD34+ expression. Non-LICs from leukemic mice mice were characterized by depletion cocktail-, c-kit-, and CD34- expression. Counterpart non-leukemic populations were isolated from wild-type control mice.
Rychtarcikova Z., Lettlova S., Tomkova V., Korenkova V., Langerova L., Simonova E., Zjablovskaja P., Alberich-Jorda M., Neuzil J, & Truksa J. (2016). Tumor-initiating cells of breast and prostate origin show alterations in the expression of genes related to iron metabolism. Oncotarget, 8(4), 6376-6398.
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5

Multiparameter Flow Cytometry Analysis

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Cells were stained under dark for 30 min in PEB buffer containing antibodies, washed thrice with ice cold PBS and analyzed in a BD LSR II (BD Biosciences) instrument or FACS-purified with a BD FACS Aria II or a SH800S (Sony) cell sorter. Dead cells and doublets were excluded on the basis of FSC and SSC distribution and DAPI (Sigma) exclusion. Antibodies used were: B220 (clone RA3–6B2), CD3 (clone 145–2C11), CD8 (clone 53–6.7), CD11b (clone M1/70), CD11c (clone N418), CD16/32 (clone 93), CD24 (clone 30-F1), CD31 (clone A20), CD41 (clone MWReg30), CD45 (clone 30-F11), CD45.1 (clone A20), CD45.2 (clone 104), CD48 (clone HM48–1), CD105 (clone MJ7/18), CD115 (clone AFS98), CD135 (clone A2F10), CD144 (clone BV13), CD150 (clone TC15–12F12.2), CD169 (clone 3D6.112), CD172α (clone P84), F4/80 (clone BM8), Gr1 (clone RB6–8C5), Ly6C (clone HK1.4), Ly6-G (clone 1A8), Sca-1 (clone D7), Ter119 (clone TER-119), MHC II (clone M5/114.15.2), from BioLegend; CD34 (clone RAM34), CD117 (clone 2B8), and Siglec-H (clone eBio440c), from BioLegend or Thermo Fisher Scientific. Data was analyzed with FlowJo (Tree Star). Gating strategies for most analyses are shown in the main or Extended Data Figures. CDP33 , pre-DC35 , cDC1 and cDC235 , CMP36 and macrophages37 are gated as described.
Zhang J., Wu Q., Johnson C.B., Pham G., Kinder J.M., Olsson A., Slaughter A., May M., Weinhaus B., D’Alessandro A., Douglas Engel J., Jiang J.X., Kofron J.M., Huang L.F., Surya Prasath V.B., Sing Way S., Salomonis N., Grimes H.L, & Lucas D. (2021). In situ mapping identifies distinct vascular niches for myelopoiesis. Nature, 590(7846), 457-462.
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6

Multiparameter Flow Cytometry Analysis

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Cells were stained under dark for 30 min in PEB buffer containing antibodies, washed thrice with ice cold PBS and analyzed in a BD LSR II (BD Biosciences) instrument or FACS-purified with a BD FACS Aria II or a SH800S (Sony) cell sorter. Dead cells and doublets were excluded on the basis of FSC and SSC distribution and DAPI (Sigma) exclusion. Antibodies used were: B220 (clone RA3–6B2), CD3 (clone 145–2C11), CD8 (clone 53–6.7), CD11b (clone M1/70), CD11c (clone N418), CD16/32 (clone 93), CD24 (clone 30-F1), CD31 (clone A20), CD41 (clone MWReg30), CD45 (clone 30-F11), CD45.1 (clone A20), CD45.2 (clone 104), CD48 (clone HM48–1), CD105 (clone MJ7/18), CD115 (clone AFS98), CD135 (clone A2F10), CD144 (clone BV13), CD150 (clone TC15–12F12.2), CD169 (clone 3D6.112), CD172α (clone P84), F4/80 (clone BM8), Gr1 (clone RB6–8C5), Ly6C (clone HK1.4), Ly6-G (clone 1A8), Sca-1 (clone D7), Ter119 (clone TER-119), MHC II (clone M5/114.15.2), from BioLegend; CD34 (clone RAM34), CD117 (clone 2B8), and Siglec-H (clone eBio440c), from BioLegend or Thermo Fisher Scientific. Data was analyzed with FlowJo (Tree Star). Gating strategies for most analyses are shown in the main or Extended Data Figures. CDP33 , pre-DC35 , cDC1 and cDC235 , CMP36 and macrophages37 are gated as described.
Zhang J., Wu Q., Johnson C.B., Pham G., Kinder J.M., Olsson A., Slaughter A., May M., Weinhaus B., D’Alessandro A., Douglas Engel J., Jiang J.X., Kofron J.M., Huang L.F., Surya Prasath V.B., Sing Way S., Salomonis N., Grimes H.L, & Lucas D. (2021). In situ mapping identifies distinct vascular niches for myelopoiesis. Nature, 590(7846), 457-462.
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7

Bone Marrow Cell Cycle Analysis

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Bone marrow cells were analyzed for cell cycle phases after lineage depletion and surface staining, followed by fixation and permeabilization and staining with DAPI (4-6-diamidino-2-phenylindole, dihydrochloride, Molecular Probes). Lin− cells were obtained by blocking the FcγIII/II receptor using antibody clone 2.4 G2 and then incubated with biotinylated antibodies against Mac-1/CD11b (clone M1/70), B220 (RA3-6B2), Gr-1 (RB6-8C5), CD3ε (500A2), and Ter-119 (Ter-119) followed by streptavidin microbeads (Miltenyi Biotec). The cells were passed through magnetic columns (Miltenyi Biotec) to collect the lineage-depleted cells, and surface staining was done with fluorescently labeled stem cell antigen (Sca1) (clone D7, Biolegend) and stem cell factor (ckit) (288, BD) antibodies. The cells were cytofixed and permeabilized (BD cytofix/cytoperm) and then stained with 1 mg/mL DAPI at room temperature for thirty minutes. Cells were centrifuged and resuspended in PBS before acquisition on LSR II. Doublets were eliminated and single cells were considered for LSK (Lin−Sca1+cKit+) population that was analyzed for cell cycle phases based on differential staining ability.
Unnisa Z., Singh K.P., Henry E.C., Donegan C.L., Bennett J.A, & Gasiewicz T.A. (2016). Aryl Hydrocarbon Receptor Deficiency in an Exon 3 Deletion Mouse Model Promotes Hematopoietic Stem Cell Proliferation and Impacts Endosteal Niche Cells. Stem Cells International, 2016, 4536187.
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