Blood collection began 4 weeks post-transplant through retro-orbital puncture, collected into EDTA Microtainers (BD Biosciences, San Jose, CA, USA), and stained as previously described.27 (link) Antibodies used were mouse CD45.1/CD45.2-V500 (30-F11), CD3-Pacific Blue (17A2), CD4-allophycocyanin (APC) (RM4-5), CD8-peridinin-chlorophyll-protein (PerCP) (53-6.7), CD14-phycoerythrin (PE) (rmC5-3), CD19-APC-H7 (1D3), Lineage-V450, c-Kit-APC (2B8), Sca1-PE-Cy7 (D7), CD150-PerCP/Cy5.5 (TC15-12F12.2), and CD48-APC-Cy7 (HM48-1) (BD Biosciences, BioLegend [San Diego, CA, USA], eBioscience [Waltham, MA, USA]). Lineage subsets were gated based on side scatter with CD45 on the x axis (Figure S4 ). Lymphocyte subsets were further analyzed based on surface markers (Figure S4 ). DNA was extracted (QIAGEN, Hilden, Germany) and analyzed as previously described.23 (link)
Sca 1 pe cy7 d7
Manufactured by BD
Sourced in United States
Sca-1-PE-Cy7 (D7) is a fluorescently-labeled antibody used for flow cytometry analysis. It targets the Sca-1 (Stem cell antigen-1) cell surface marker, which is commonly expressed on hematopoietic stem and progenitor cells. The antibody is conjugated with the fluorescent dyes Phycoerythrin (PE) and Cyanine 7 (Cy7), allowing for multicolor flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Efluor 450, by BD (2 mentions)
C kit apc eflour780 2b8, by BD (2 mentions)
Cd150 alexa fluor 647 tc15 12ff12, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Edta microtainers, by BD (1 mentions)
C kit apc 2b8, by BD (1 mentions)
Cd138 pe, by BD (1 mentions)
Cd135 pe, by BD (1 mentions)
Cd41 fitc, by Thermo Fisher Scientific (1 mentions)
Cd150 pe, by Thermo Fisher Scientific (1 mentions)
Cd34 fitc ram34, by Thermo Fisher Scientific (1 mentions)
4 protocols using sca 1 pe cy7 d7
1
Multidimensional Immune Profiling Post-Transplant
2
Multiparameter FACS Analysis of Hematopoietic Stem Cells
3
Bone Marrow Cell Immunophenotyping Protocol
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Six weeks after tumour cell injection, mice were euthanized humanely. The femurs, tibias, and iliac crest were harvested immediately and placed in PBS. Bone marrow cells were obtained by rinsing femurs, tibias, and iliac crest with PBS. After collection, 1 × 106–5 × 106 cells were suspended in 100 μl of FACS buffer and stained with the appropriate antibodies at 4 °C. According to the manufacturer’s protocol, 10 μl of each antibody was sequentially added to the cell suspension on ice. The workflow is shown in Supplementary Figure 1 . Fresh BM cells were stained with the following antibodies: lineage antibodies (B220 (RA3-6B2), CD3 (17A2), Gr-1 (RB6-8C5), CD11b (M1/70), CD8a (53–6.7), CD4 (GK1.5) and Ter-119 (TER-119)), Sca-1-Pe-Cy7 (D7, BD Biosciences), c-Kit-APC (2B8), CD150-PE (TC15-12F12.2), CD48-PerCP (HM48-1, eBioscience), CD41-FITC (MWReg30), CD34-FITC (RAM34, eBioscience), CD16/32-BV421 (93), CD71-FITC (C2, BD Biosciences), CD135-PE (A2F10) and CD138-PE (281-2).
4
Multiparameter FACS Analysis of Hematopoietic Stem Cells
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Multiparameter FACS analyses were performed to determine populations of HSCs (Lineage−Sca-1+c-Kit+Flk2−CD48−CD150+), multipotent progenitor sub-population 1 (MPP1, Lineage−Sca-1+c-Kit+Flk2−CD48−CD150−), MPP2 (Lineage−Sca-1+c-Kit+Flk2−CD48+CD150+), and MPP3 (Lineage−Sca-1+c-Kit+Flk2−CD48+CD150−). Antibodies were purchased from eBiosciences, San Diego, CA, unless otherwise noted. BM cells freshly harvested from femurs and tibias were stained with biotin-labeled antibodies against mouse hematopoietic lineage markers: Mac-1 (M1/70), Gr-1 (RB6-8C5), Ter119 (TER-119), CD3e (145-2C11), B220 (RA3-6B2), and subsequently stained with antibodies conjugated with various fluorochromes: streptavidin eFluor® 450, Sca-1-PE-Cy7 (D7, BD Biosciences, San Jose, CA), c-Kit-APC-eFlour780 (2B8), CD150-Alexa Fluor® 647 (TC15-12FF12.2, BD Biosciences), CD48-FITC (HM48-1), Flk2-PE (A2F10.1, BD Biosciences) for HSCs or MPPs. Specific cell populations were gated based on immune phenotypes for quantification or cell sorting. Fluorescence Minus One (FMO) was used for setting the gating on control samples. Flow cytometric analyses were performed on BD LSR II (BD Biosciences, San Jose). Cell sorting was conducted using BD FACSAria. Data were analyzed using FlowJo software (TreeStar, Ashland).
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