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Sca 1 fitc

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The Sca-1-FITC is a fluorescently-labeled antibody that targets the Sca-1 (Stem Cell Antigen-1) surface marker. It is commonly used in flow cytometry applications to identify and analyze stem and progenitor cells in various sample types.

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Lab products found in correlation

Propidium iodide staining solution, by Merck Group (2 mentions) Collagenase, by Merck Group (2 mentions) Cd31 pe cy7, by Thermo Fisher Scientific (2 mentions) F4 80 apc cy7, by BioLegend (2 mentions) F4 80 apc, by Thermo Fisher Scientific (2 mentions) Sytox blue, by Thermo Fisher Scientific (2 mentions) Cd45 pe cy7, by Thermo Fisher Scientific (2 mentions) Cd206 alex647, by Bio-Rad (2 mentions) Sytox blue, by Tree Star (2 mentions) Facsaria cell sorter, by BD (2 mentions) Cd11c pe, by BD (2 mentions) Cd90 fitc, by BD (2 mentions) Cd73 pe, by BD (2 mentions) Cd45 efluor450, by Thermo Fisher Scientific (2 mentions) Cd31 efluor450, by Thermo Fisher Scientific (2 mentions) Ter119 efluor450, by Thermo Fisher Scientific (2 mentions) Collagenase a, by Roche (2 mentions) Cd45 pe, by Thermo Fisher Scientific (2 mentions) Streptavidin percp cy5, by BD (1 mentions) Cd16 cd32, by Thermo Fisher Scientific (1 mentions)

12 protocols using sca 1 fitc

1

Hematopoietic Lineage Analysis in Knockout Mice

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For analysis of hematopoietic lineage markers in the BMs of mice with specific knockout of Wls or Six1 in hematopoietic cells, we follow protocols described previously [30 (link)]. Characterization of MLL-AF9 AML and isolation of the enriched LIC population from BMs of AML mice have been described previously [28 (link)]. Fluorescein-labeled antibodies used in the study include: Biotin Mouse Lineage Depletion Cocktail (BD Biosciences); Streptavidin-PerCP-Cy5.5, Sca1-FITC, cKit-APC-Cy7, cKit-PE, FcγR-PE-Cy7, CD34-PacificBlue, FLT3-PE, IL7R-APC, Mac1-APC, B220-PE, CD3-APC, Gr1-PE, Mac1-APC, CD16/CD32, all antibodies are anti-mouse and were purchased from eBiosciences.
Zhang L.S., Kang X., Lu J., Zhang Y., Wu X., Wu G., Zheng J., Tuladhar R., Shi H., Wang Q., Morlock L., Yao H., Huang L.J., Maire P., Kim J., Williams N., Xu J., Chen C., Zhang C.C, & Lum L. (2018). Installation of a cancer promoting WNT/SIX1 signaling axis by the oncofusion protein MLL-AF9. EBioMedicine, 39, 145-158.
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2

Flow Cytometric Analysis of Adipose Tissue

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Stromal vascular fraction (SVF) was obtained from pgWAT by treatment with 2 mg/mL collagenase (Sigma) for 45 min at 37 °C. The isolated SVF was resuspended in cold Hank’s balanced salt solution (HBSS) with 2% fetal bovine serum (FBS). Cells were incubated with CD45-PE-Cy7 (eBiosciences), F4/80-APC-Cy7 (BioLegend), CD206-Alex647 (Serotec, Inc.) and CD11c-PE (BD Pharmingen) antibodies for 30 min in HBSS containing 2% FBS on ice and then washed and resuspended in solution with Sytox Blue (Thermo Scientific). Cells were analyzed on a BD FACSAria cell sorter after selection by forward scatter and side scatter, followed by exclusion of dead cells with Sytox Blue staining, and analyzed for cell-surface markers using FlowJo software (Tree Star). M1 or M2 macrophages were identified as F4/80-positive/CD11c-positive/CD206-negative or F4/80-positive/CD11c-negative/CD206-positive cells, respectively. The data are shown as the percentage of M1 and M2 macrophages. For sorting preadipocytes, endothelial cells and macrophages, cells were incubated with CD31-PE-Cy7 (eBiosciences), F4/80-APC (eBiosciences), and Sca-1 FITC (eBiosciences) antibodies for 30 min in HBSS containing 2% FBS on ice and then washed and resuspended in solution with Propidium Iodide Staining Solution (Sigma). Gating strategies are presented in Supplementary Figure 12 and Supplementary Figure 13.
Takahashi H., Alves C.R., Stanford K.I., Middelbeek R.J., Pasquale N.i.g.r.o., Ryan R.E., Xue R., Sakaguchi M., Lynes M.D., So K., Mul J.D., Lee M.Y., Balan E., Pan H., Dreyfuss J.M., Hirshman M.F., Azhar M., Hannukainen J.C., Nuutila P., Kalliokoski K.K., Nielsen S., Pedersen B.K., Kahn C.R., Tseng Y.H, & Goodyear L.J. (2019). TGF-β2 is an exercise-induced adipokine that regulates glucose and fatty acid metabolism. Nature metabolism, 1(2), 291-303.
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3

Flow Cytometric Analysis of Adipose Tissue

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Stromal vascular fraction (SVF) was obtained from pgWAT by treatment with 2 mg/mL collagenase (Sigma) for 45 min at 37 °C. The isolated SVF was resuspended in cold Hank’s balanced salt solution (HBSS) with 2% fetal bovine serum (FBS). Cells were incubated with CD45-PE-Cy7 (eBiosciences), F4/80-APC-Cy7 (BioLegend), CD206-Alex647 (Serotec, Inc.) and CD11c-PE (BD Pharmingen) antibodies for 30 min in HBSS containing 2% FBS on ice and then washed and resuspended in solution with Sytox Blue (Thermo Scientific). Cells were analyzed on a BD FACSAria cell sorter after selection by forward scatter and side scatter, followed by exclusion of dead cells with Sytox Blue staining, and analyzed for cell-surface markers using FlowJo software (Tree Star). M1 or M2 macrophages were identified as F4/80-positive/CD11c-positive/CD206-negative or F4/80-positive/CD11c-negative/CD206-positive cells, respectively. The data are shown as the percentage of M1 and M2 macrophages. For sorting preadipocytes, endothelial cells and macrophages, cells were incubated with CD31-PE-Cy7 (eBiosciences), F4/80-APC (eBiosciences), and Sca-1 FITC (eBiosciences) antibodies for 30 min in HBSS containing 2% FBS on ice and then washed and resuspended in solution with Propidium Iodide Staining Solution (Sigma). Gating strategies are presented in Supplementary Figure 12 and Supplementary Figure 13.
Takahashi H., Alves C.R., Stanford K.I., Middelbeek R.J., Pasquale N.i.g.r.o., Ryan R.E., Xue R., Sakaguchi M., Lynes M.D., So K., Mul J.D., Lee M.Y., Balan E., Pan H., Dreyfuss J.M., Hirshman M.F., Azhar M., Hannukainen J.C., Nuutila P., Kalliokoski K.K., Nielsen S., Pedersen B.K., Kahn C.R., Tseng Y.H, & Goodyear L.J. (2019). TGF-β2 is an exercise-induced adipokine that regulates glucose and fatty acid metabolism. Nature metabolism, 1(2), 291-303.
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4

Characterization of Mesenchymal Stem Cells

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After 10 passages, the cells were characterized by multiple commonly utilized markers for conventional MSCs. Phenotypic antibodies used are as follows: CD45-FITC, CD90-FITC, CD34-PE, CD11c-FITC, CD44-PE, I-Ab-FITC, CD80-FITC, CD11b-PE-Cy7, CD73-PE (all from BD Biosciences) and H-2kb-APC, CD105-PE, Sca-1-FITC, CD9-PE, and CD86-PE (all from eBioscience). All isotype controls were purchased from BioLegend. Flow Cytometry was conducted on a BD FacsCalibur. Standard protocols were conducted to verify the ability of MSCs to differentiate into adipocytes and osteoblasts using Mesencult mouse adipogenic stimulatory supplements and mouse osteogenic stimulatory supplements (StemCell Technologies products), respectively, in IMDM-based media with 10% FBS supplementation. Supplement-to-basal media was at a 1:4 ratio. For both differentiation tests, 2.5×104 MSC were plated per well in 2mL Mesencult media in 6-well plates in 37°C/ 10% CO2 incubation. On day 3, cells were given appropriate differentiation media, with media changes every 3– 4 days. After 2 weeks of culture, cells were stained for adipocytic differentiation with Oil Red O and osteogenic differentiation with Alizarin red. Human MSCs were phenotyped by Lonza as CD105+CD166+CD29+CD44+ > 90% and CD14+CD34+CD45+ <10%.
Glenn J.D., Smith M.D., Calabresi P.A, & Whartenby K.A. (2014). Mesenchymal stem cells differentially modulate effector CD8+ T cell subsets and exacerbate experimental autoimmune encephalomyelitis. Stem cells (Dayton, Ohio), 32(10), 2744-2755.
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5

Immunofluorescence Analysis of Muscle Stem Cells

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Primary antibodies used for immunofluorescence were against MYOD (1:20; Santa Cruz Biotechnology), MYHC (1:20; MF-20, DSHB), PERILIPIN A/B (1:100; Sigma-Aldrich), Sca-1-FITC (1:100; eBioscience), and α7-Integrin-PE (1:100; eBioscence). Oil red O was used to stain lipids, as described in Mozzetta et al. (2013) (link). Further experimental details are provided in the Supplemental Material.
Saccone V., Consalvi S., Giordani L., Mozzetta C., Barozzi I., Sandoná M., Ryan T., Rojas-Muñoz A., Madaro L., Fasanaro P., Borsellino G., De Bardi M., Frigè G., Termanini A., Sun X., Rossant J., Bruneau B.G., Mercola M., Minucci S, & Puri P.L. (2014). HDAC-regulated myomiRs control BAF60 variant exchange and direct the functional phenotype of fibro-adipogenic progenitors in dystrophic muscles. Genes & Development, 28(8), 841-857.
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6

Isolation of Murine Muscle Satellite Cells

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Tibialis anterior muscles were subjected to enzymatic dissociation in PBS (Cat #14040-091; Gibco) with 2 mg/ml collagenase A (Cat # 10 103 586 001; Roche), 2.4 U/ml Dispase I (Cat # 04 942 078 001; Roche), 10 ng/ml DNase (Cat #11 284 932 001; Sigma-Aldrich), 0.4 mM CaCl 2 , and 5 mM MgCl 2 for 60 min at 37°C under gentle agitation. The supernatants were filtered through a 100-and 40µm cell strainers (#08-771-19, #08-771-1; BD Falcon) and incubated with the following antibodies for 30 min on ice: CD45-eFluor 450 (1/50, #48-0451-82; eBioscience), CD31-eFluor 450 (1/50, #48-0311-82; eBioscience), TER-119-eFluor 450 (1/50, #48-5921-82; eBioscience), Sca1-FITC (1/50, Ly-6A/E FITC, clone D7, #11-5981-82; eBioscience), Itga7-649 (1/500, #67-0010-01; AbLab). MuSCs were isolated as TER-119-/CD45-/CD31-/Itga7+/SCA-1-cells. Isolated satellite cells were used either for RNA extraction and WB analysis, or plated on glass slides (177402; Thermo Fisher Scientific) for immunostaining analysis.
Catarinella G., Bracaglia A., Skafida E., Procopio P., Ruggieri V., Parisi C., De Bardi M., Borsellino G., Madaro L., Puri P.L., Sacco A, & Latella L. (2024). STAT3 inhibition recovers regeneration of aged muscles by restoring autophagy in muscle stem cells. Life science alliance, 7(8).
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7

Immunophenotypic Characterization of MSCs

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Prior to analysis and sorting by flow cytometry, distinct amounts of MSCs were stained with fluorochrome-conjugated antibodies. Antibody specifications and concentrations used were the following: CD13-PE (BD Biosciences, 347406), CD45-PE (eBioscience, 12-0451-81), Ter-119-PE (eBioscience, 12-5921-81), Sca-1-FITC (eBioscience 11-5981-81), PDFGRα-APC (BD Biosciences, 562777), CD44-FITC (BD Biosciences, 347943), CD19-PerCP (BD Biosciences, 332780), CD90-FITC (BD Biosciences, 555595), HLA-DR-PerCP (BD Biosciences, 347402), CD14-FITC (BD Biosciences, 345784), CD166-PE (BD Biosciences, 559263), CD45-PErcCPcy5.5 (BD Biosciences, 332784), CD34-FITC (Invitrogen, 11-0349-42), CD73-PE (BD Biosciences, 550257), CD105-APC (R&D System, FAB10971A).
Garcia-Gomez A., Li T., de la Calle-Fabregat C., Rodríguez-Ubreva J., Ciudad L., Català-Moll F., Godoy-Tena G., Martín-Sánchez M., San-Segundo L., Muntión S., Morales X., Ortiz-de-Solórzano C., Oyarzabal J., San José-Enériz E., Esteller M., Agirre X., Prosper F., Garayoa M, & Ballestar E. (2021). Targeting aberrant DNA methylation in mesenchymal stromal cells as a treatment for myeloma bone disease. Nature Communications, 12, 421.
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8

Isolation of Fibro-Adipogenic Progenitors from mdx Mice

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FAPs were isolated from C57Bl6 mdx mice at the end of the treatments immediately after the sacrifice. Hind limb muscles for each mouse were minced and put into a 15 ml tube containing 4 ml of digest solution in HBSS (#24020-091,GIBCO) containing 2 mg/ml Collagenase A (#10103586001, Roche), 2.4 U/ml Dispase II (#04942078001, Roche), 10 ng/ml DNase I (#11284932001, Roche) for 90 min at 37°C. Cells were filtered through 100um, 70um and 40um cell strainers (#08-771-19, #08-771-2, #08-771-1, BD Falcon) and resuspended in 0.5 ml of HBSS containing 0.2% w/v BSA and 1% v/v Penicillin-Streptomycin for the staining of cell surface antigens 30 min on ice. The following antibodies were used: CD45-eFluor 450 (1:50, #48-0451-82, eBiosciences), CD31-eFluor 450 (1:50, #48-0311-82, eBioscience), Ter119-eFluor 450 (1:50, #48-5921-82, eBiosciences), Itga7-649 (1:500, #67-0010-01, AbLab) and Sca1-FITC (1:50, 5981-82, eBioscience). Cells were finally washed and resuspended in 1 ml of HBSS containing 0.2% w/v BSA and 1% v/v Penicillin-Streptomycin. FAPs were isolated as Ter119 -/CD45 -/CD31 -/ Itga7 -/Sca1 + cells using a Beckman Coulter MoFlo Legacy high-speed cell sorter.
Consalvi S., Tucciarone L., Macrì E., Bardi M.D., Picozza M., Salvatori I., Renzini A., Valente S., Mai A., Moresi V., & Puri P. (2021). Partial resistance to HDAC inhibitors in FAPs of dystrophic muscles at late stages of disease is associated to epigenetic and transcriptional features of cellular senescence.
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9

Comprehensive Cell Sorting and Immunodetection Protocol

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For cell sorting, CD140a-APC (#17140181), Sca-1-FITC (#11598185), Terr-119-PE (#12592182) and the Fixable Viability Dye eFluor 450 (#65086314) were all purchased from eBioscience, whereas CD45-PE (#A16325) antibody was obtained from Life Technologies.
For immunofluorescence and Western Blot experiments, antibodies against acetyl-Lysine (#9441S), Fatty Acid Synthase (#3189S), CBP (7389S), ACS (#3658T), anti-rabbit IgG HRPlinked (#7074S), anti-mouse IgG HRP-linked (#7076S) and histone H3 (#14269) were all purchased from Cell Signaling Technology. Antibodies against ACC1 (#21923-1-AP), ACLY (#15421-1-AP), KAT2A/GCN5 (14983-1-AP) and Citrate carrier (#15235-1-AP) were bought from ProteinTech. TOMM20 antibody (WH0009804M1) was obtained from Sigma-Aldrich.
histone H3 acetylation (#39139) and H3K27 acetylation (#39133) antibodies were all obtained from Active Motif. β-actin antibody (sc-47778) was bought from Santa Cruz Biotechnology.
Anti-Mouse IgG Alexa fluor 488 (#A11001), anti-Rabbit IgG Alexa Fluor 488, anti-Rabbit IgG Alexa fluor 594 (#A-11012) were all bought from ThermoFisher Scientific.
Pouikli A., Maleszewska M., Parekh S., Nikopoulou C., Bonfiglio J.J., Mylonas C., Sandoval T., Schumacher A., Hinze Y., Matić I., & Tessarz P. (2022). Hypoxia promotes osteogenesis via regulation of the mito-nuclear communication.
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10

Flow Cytometry Analysis of Bone Cells

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Cells were collected from hindlimb bone tissue as described in “Flow cytometry and single-cell RNA-seq,” resuspended in buffer solution (PBS + 2% FBS + 2 mM EDTA), and blocked with recombinant Fc protein (1:100 dilution, BD Biosciences, catalog no. 553142). Cells were then incubated with the primary antibody solution (1:100 dilution; PDGFRA-APC, Thermo Fisher Scientific, catalog no. 17-1401-81; SCA1-FITC, Thermo Fisher Scientific, 11-5981-82; CD45-BV450, BD Biosciences, catalog no. 560697; TER119-BV450, BD Biosciences, catalog no. 560504; CD31-Pacific Blue, BioLegend [San Diego, CA, USA], 102422; DAPI, Sigma, catalog no. 10236276001) for 30 minutes on ice, washed with buffer solution, and analyzed using the BD FACS-Canto system (BD Biosciences). Specimens were analyzed in trip-licate with at least 100,000 events per replicate.
Vesprey A., Suh E.S., Aytürk D.G., Yang X., Rogers M., Sosa B., Niu Y., Kalajzic I., Ivashkiv L.B., Bostrom M.P, & Ayturk U.M. (2021). Tmem100- and Acta2-Lineage Cells Contribute to Implant Osseointegration in a Mouse Model. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 36(5), 1000-1011.
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