Sca 1 pacific blue

Manufactured by BioLegend

The Sca-1-Pacific blue is a fluorochrome-conjugated antibody used for flow cytometry analysis. It binds to the Sca-1 (Stem cell antigen-1) surface marker, which is expressed on various hematopoietic and non-hematopoietic stem and progenitor cells. The Pacific blue fluorescent dye is used for conjugation, providing a specific emission profile suitable for multicolor flow cytometry applications.

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Lab products found in correlation

Propidium iodide, by Thermo Fisher Scientific (3 mentions) Cd115 afs98 apc, by Thermo Fisher Scientific (2 mentions) Cd45 30 f11 apccy7, by Thermo Fisher Scientific (2 mentions) Ly6c g or gr 1 rb6 8c5 percpcy5, by Thermo Fisher Scientific (2 mentions) Facsaria 2, by BD (2 mentions) C kit apc, by BD (1 mentions) Trucount control beads, by BD (1 mentions) Lsrii fortessa, by BD (1 mentions) Ter119, by Thermo Fisher Scientific (1 mentions) Cd34 apc, by BioLegend (1 mentions) Cd34 efluor 660, by Thermo Fisher Scientific (1 mentions) Facsaria fusion sorter, by BD (1 mentions) Itga6 pe cy7, by BioLegend (1 mentions) Facscanto 2, by BD (1 mentions) Cd105 apc, by BioLegend (1 mentions) Magnisort beads, by Thermo Fisher Scientific (1 mentions) Facsaria 3, by BD (1 mentions) Ter119, by BioLegend (1 mentions) C kit apccy7, by BioLegend (1 mentions) Slam pe cy7, by BioLegend (1 mentions)

Spelling variants of this product

Sca-1 (56 citations) Ly-6A/E (Sca-1; Pacific Blue (PB) (2 citations) Sca-1-Pacific Blue (clone D7; (2 citations)

8 protocols using sca 1 pacific blue

Quantifying Hematopoietic Stem Cell Clones

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Clone size calculations were determined as previously described [12] (link). After 10 days of culture, single HSC-derived clones were estimated to be small (50–5,000 cells), medium (5,000–20,000 cells), or large (≥20,000). No clones had fewer than 50 cells. Ten-day clones were stained with biotinylated lineage marker antibodies (Haematopoietic Progenitor Enrichment Cocktail, STEMCELL), c-Kit-APC (BD) and Sca-1-Pacific Blue (Biolegend). To enumerate cells, a defined number of fluorescent beads (Trucount Control Beads, BD) were added to each well, and each sample was backcalculated to the proportion of the total that were run through the cytometer. Small clones were not able to be assessed individually by flow cytometry and were pooled; the percentage of c-Kit⁺Sca⁺Lin⁻ (KSL) cells was greater than 95%. Flow cytometry was performed on an LSRII Fortessa (BD), and all data were analyzed using Flowjo 10.0.7 (Treestar, USA).

Schulte R., Wilson N.K., Prick J.C., Cossetti C., Maj M.K., Gottgens B, & Kent D.G. (2015). Index sorting resolves heterogeneous murine hematopoietic stem cell populations. Experimental Hematology, 43(9), 803-811.

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Analyzing Hematopoietic Stem Cell Progenitors

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Hematopoietic stem cell precursor cells (HSPCs) and myeloid progenitor cells including common myeloid progenitor cells (CMPs) and granulocyte-monocyte progenitor cells (GMPs) from bone marrow were analyzed by flow cytometry as described in Figure S2D. Bone marrow was harvested from femurs and tibias by flushing bones with cold Hank’s balanced salt solution (HBSS) containing 0.1% BSA and 2mM EDTA. RBC lysis was performed once for 5 minutes, followed by antibody staining with a cocktail of antibodies against lineage-committed cells (CD3e, CD19, CD45R (B220), CD11b, TER-119, CD2, CD8b, CD4, Ly6G; all FITC-labeled and from eBioscience), Sca-1 Pacific Blue, c-kit (CD117) APC-Cy7, CD34 APC, CD16/CD32 (FcyRII/III) PerCp-Cy5.5 (all from BioLegend). HSPCs were identified as lin- Sca-1⁺ c-kit⁺; CMPs as lin⁻ Sca1⁻ c-kit⁺ CD34^int FcγRII/III^int; GMPs as lin⁻ Sca1⁻ c-kit⁺ CD34^int FcγRII/III^hi. Isolated and stained bone marrow cells were either directly analyzed by LSRII (BD), or flow sorted by FACS Aria II for further RNA isolation and RNA/ATAC-sequencing, or in vitro assays.

Christ A., Günther P., Lauterbach M.A., Duewell P., Biswas D., Pelka K., Scholz C.J., Oosting M., Haendler K., Baßler K., Klee K., Schulte-Schrepping J., Ulas T., Moorlag S.J., Kumar V., Park M.H., Joosten L.A., Groh L.A., Riksen N.P., Espevik T., Schlitzer A., Li Y., Fitzgerald M.L., Netea M.G., Schultze J.L, & Latz E. (2018). Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming. Cell, 172(1-2), 162-175.e14.

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Isolation and Purification of Keratinocytes

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Matrigel droplets were degraded in 0.5% trypsin, 0.5 mM EDTA in PBS for 8 min at 37°C. Trypsin was neutralized using cold KGM. After centrifugation for 5 min at 600 g, cells were washed with FACS buffer (2% FBS, 2 mM EDTA in PBS). Surface marker staining was performed for 30 min on ice with the following antibodies: CD34-eFluor 660 (eBioscience, clone RAM34, 1:100) and ITGA6-PE/Cy7 (Biolegend, clone GoH3, 1:1000) for sorting or CD34-eFluor 660 (eBioscience, clone RAM34, 1:100) and ITGA6-Pacific Blue (Biolegend, clone GoH3, 1:200) for analysis. Freshly isolated keratinocytes were stained additionally with Sca1-Pacific Blue (Biolegend, clone D7, 1:400). 7AAD or propidium iodide (PI) was used to exclude dead cells. Cells were analyzed on FACSCantoII (BD Biosciences) or sorted on FACSAria IIIu Svea and FACSAria Fusion sorters (BD Biosciences). Sorted cells were collected in 15 ml tubes containing KGM at 4°C.

Allmeroth K., Kim C.S., Annibal A., Pouikli A., Koester J., Derisbourg M.J., Andrés Chacón-Martínez C., Latza C., Antebi A., Tessarz P., Wickström S.A, & Denzel M.S. (2021). N1-acetylspermidine is a determinant of hair follicle stem cell fate. Journal of Cell Science, 134(9), jcs252767.

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Hematopoietic Lineage Depletion and FACS Sorting

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BM suspensions were prepared as described above, and lineage-positive cells were depleted using a lineage depletion kit (eBioscience mouse hematopoietic lineage biotin panel; ThermoFisher Scientific) according to the manufacturer’s instructions. Briefly, BM derived from the femurs and tibiae of three mice was incubated with biotinylated antibodies against CD3, B220, Gr1, and Ter119 in a volume of 1 ml PBS/0.1% BSA for 10 min at 4°C. Cells were then washed and incubated with 0.4 ml streptavidin bound to magnetic beads (Magnisort beads; ThermoFisher Scientific) in a volume of 2 ml PBS/0.1% BSA in a FACS tube for 10 min at room temperature. The volume was then made up to 4 ml, and the FACS tube was placed inside a magnet for 10 min at room temperature. The fraction of cells not bound to the magnet was poured out and stained for FACS as described above.
Pre–CFU-Es and CFU-Es were sorted by FACS using a FACS ARIA III (BD) with 70-µm nozzle. For culture, cells were sorted into 0.5 ml IMDM medium with 5% FCS in a 5-ml FACS tube. For RT quantitative PCR (RT-qPCR), cells were sorted into 350 µl RLT buffer with β-mercaptoethanol in 1.5 ml tubes. The following staining panel was used for sorting: Sca-1-Pacific blue, PE-CD117, APC-CD105, PerCP Cy5.5-CD11b and Streptavidin (BioLegend), APC Cy7-CD16/32, PE Cy7-CD41, BV605-CD150.

Swann J.W., Koneva L.A., Regan-Komito D., Sansom S.N., Powrie F, & Griseri T. (2020). IL-33 promotes anemia during chronic inflammation by inhibiting differentiation of erythroid progenitors. The Journal of Experimental Medicine, 217(9), e20200164.

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Comprehensive Immune Phenotyping Protocol

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TCR-β (H57–597), F4/80 (BM8), CD2 (RM2–5), CD3e (145–2C11), CD4 (GK1.5), CD8b (53–6.7), CD19 (eBio1D3), CD45R (B220, RA3–6B2), Gr-1 (Ly6G, RB6–8C5), Cd11b (Mac1, M1/70), Ter119 (Ly76) and NK1.1 (Ly53, PK136)-FITC were all from eBioscience and used for lineage determination. c-Kit (CD117, ACK2)-APCeFluor780 from eBioscience, Sca-1-Pacific blue from Biolegend, FcgRII/lll-PE (CD 16/32, 2.4G2), CD34 (RAM34)-AlexaFluor 647, CD135 (Flt3, A2F10)-PE, CD150 (Slamfl, TC15–12F12.2)-PECy7 were from Biolegend and used to quantify HSPCs and progenitor subsets. Peripheral leukocytes were stained with CD115 (AFS98)-APC, CD45 (30-F11)-APCCy7 and Ly6C/G or Gr-1 (RB6–8C5)-PercPCy5.5 from eBioscience and BD Biosciences, respectively.

Viaud M., Abdel-Wahab O., Gall J., Ivanov S., Guinamard R., Sore S., Merlin J., Ayrault M., Guilbaud E., Jacquel A., Auberger P., Wang N., Levine R.L., Tall A.R, & Yvan-Charvet L. (2020). ABCA1 Exerts Tumor-Suppressor Function in Myeloproliferative Neoplasms. Cell reports, 30(10), 3397-3410.e5.

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Isolation and FACS Analysis of Hematopoietic Cells

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Femora, tibiae and ilia were isolated and gently crushed in staining medium (PBS + 5mM EDTA + 2% fetal bovine serum). Unless stated otherwise, for each untreated (−) sample, the bone marrow of 3 mice were pooled, and for each pI:pC sample the bone marrow of 2 mice were pooled, to increase sample size and to decrease artefacts based on mouse-to-mouse variability. Red blood cells were lysed using Ammonium-Chloride-Potassium (ACK) lysing buffer for 1min on ice. CD117 (c-KIT) positive cells were enriched using CD117 MicroBeads (Miltenyi Biotec) and subsequently immunostained using the antibodies listed here below. Propidium Iodide (ThermoFisher Scientific) was used for negative selection of dead cells. All sorting experiments were performed using BD FACS Aria II or III flow cytometer (BD Biosciences), using the gating strategy illustrated in Figure S1A. Antibodies used for FACS sorting of haematopoietic bone marrow cells: CD3 (Biolegend), CD4 (Biolegend), CD5 (Biolegend), CD8 (Biolegend), Gr1 (Biolegend), CD11b (Biolegend), Ter119 (Biolegend, B220 (Biolegend), anti-rat PeCy5 (Invitrogen), c-kit APC-Cy7 (Biolegend), Sca1 Pacific Blue (Biolegend), SLAM PeCy7 (Biolegend), Flk2 Biotin (Biolegend), CD34 FITC (Invitrogen), Streptavidine QDot 605 (Invitrogen).

Eliceiri B., Labella T., Hagino Y., Srivastava A., Schlessinger D., Pilia G., Palmieri G, & D'Urso M. (1991). Stable integration and expression in mouse cells of yeast artificial chromosomes harboring human genes.. Proceedings of the National Academy of Sciences of the United States of America, 88(6), 2179-2183.

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Comprehensive Immune Phenotyping Protocol

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Stromal Vascular Fraction Isolation and Characterization

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Antibodies used include SCA1 Pacific Blue (BioLegend, 108120), CD45 PE-Cy7 (eBioscience, 25-0421-82), CD11b APC-Cy7 (BioLegend, 101226), and CD31 APC (eBioscience, 17-0311-82). SVF was prepared as for cell culture, except that following centrifugation, the resulting cell pellet was resuspended in 1 ml ACK Lysing Buffer (Invitrogen) to remove red blood cells. After inactivation with Hank’s balanced salt solution (HBSS)/2% FBS, samples were centrifuged again and resuspended in HBSS/2% FBS for antibody labeling for flow cytometry or fluorescence-activated cell sorting (FACS). Samples were analyzed on a BD LSR Fortessa analyzer or sorted on a BD FACS Aria II. Cell gating was based on comparison with unstained and fluorescence-minus-one-stained controls. Single live cells were discriminated by forward-scatter and side-scatter analysis and 7-AAD labeling (BD Bioscience), respectively. Cells were sorted into serum-free DMEM media for gene expression analysis or into complete media for cell culture. Flow cytometry standard files were analyzed using FlowJo version 10.

Krueger K.C., Costa M.J., Du H, & Feldman B.J. (2014). Characterization of Cre Recombinase Activity for In Vivo Targeting of Adipocyte Precursor Cells. Stem Cell Reports, 3(6), 1147-1158.

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