Sca1 pe

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Sca1-PE is a fluorescently labeled antibody used for the detection and analysis of stem cell antigen-1 (Sca-1) expression in flow cytometry applications. It binds specifically to the Sca-1 cell surface marker, which is expressed on various stem and progenitor cell populations. The PE (phycoerythrin) fluorescent label allows for the visualization and quantification of Sca-1-positive cells.

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PE conjugated anti-mouse Sca1 (4 citations) PE conjugated anti-mouse Sca1 and APC conjugated anti-mouse c-Kit (2 citations) PE-Cy7-conjugated anti-mouse Sca1 (2 citations) Anti-mouse Sca1-PE (2 citations)

12 protocols using sca1 pe

Comprehensive Hematopoietic Cell Analysis Protocol

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Flow assisted cell sorting (FACS) was performed as described elsewhere (16 (link)) with the following conjugated antibodies; Mac1 PE, Gr1 FITC, B220 APC, B220 FITC, CD19 PE, surface IgM PE, CD43 FITC, CD3 PE, CD4 APC, CD8 FITC, CD71 PE, Ter119 FITC, Sca-1 PE, cKit FITC, cKit APC-Cy7, IL7Ra PECy7, CD34 FITC, CD16/32 PE (eBiosciences or BD Biosciences). StemSep Mouse Hematopoietic Progenitor Kit Lineage Positive antibody cocktail (Stem Cell Technologies, Vancouver, Canada) was used to detect lineage positive bone marrow cells, stained as previously described (16 (link)). Apoptosis assays were conducted using AnnexinV-FITC Apoptosis Detection Kit I following the manufacturer’s recommended protocol. IHC and immunoblotting were performed as previously described (49 (link)). Formalin-fixed paraffin embedded sections were stained with hematoxylin and eosin (H&E), myeloperoxidase (MPO) (A0398, DAKO), CD3 (MCA1477, AbD Serotec), B220 (553086, BD), Ter119 (116201, BioLegend). Stained slides were scanned and imaged as described elsewhere (49 (link)). For immunoblots, primary antibodies used were anti-V5 (ab9116, Abcam or R960-25, Life Technologies), and beta-actin (Cell Signaling Technology). Protein was visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific, Illinois, USA) and Amersham Hyperfilm ECL (GE Healthcare Ltd, Buckinghamshire, UK).

Gough S.M., Lee F., Yang F., Walker R.L., Zhu Y.J., Pineda M., Onozawa M., Chung Y.J., Bilke S., Wagner E.K., Denu J.M., Ning Y., Xu B., Wang G.G., Meltzer P.S, & Aplan P.D. (2014). NUP98-PHF23 is a chromatin modifying oncoprotein that causes a wide array of leukemias sensitive to inhibition of PHD domain histone reader function. Cancer discovery, 4(5), 564-577.

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Flow Cytometry Cell Sorting

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Cells were stained for 30 minutes on ice with antibodies against cKit APC (eBioscience), Sca1 PE (eBioscience), and a FITC lineage cocktail. Cells were sorted using a MoFlow Astrios (Beckman Coulter) sorter.

Yu W., Goncalves K.A., Kishikawa H., Li S., Sun G., Yang H., Vanli N., Wu Y., Jiang Y., Hu M.G., Friedel R.H, & Hu G.F. (2017). Plexin-B2 mediates physiologic and pathologic functions of angiogenin. Cell, 171(4), 849-864.e25.

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Isolation of Murine Lung Cell Types

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Murine lung tissue was digested into a single-cell suspension as previously described using dispase (Corning catalog #354235), collagenase (Roche catalog #10103578001), and DNase (Roche catalog #10104159001). When isolating AT2 cells from Sftpc^CreERT2:R26R^TdTomato mice, the animals received 200μg/gm tamoxifen (Sigma-Aldrich catalog #T55648) in corn oil (Sigma-Aldrich catalog #C8267) via gavage 3 days prior to being euthanized. Tomato+ cells were isolated using flow cytometry (MoFlow Astrios with Summit v 6.3.0.16900 software). To isolate AT2 cells from mice we stained single-cell suspensions of murine lung tissue and gated based on the following criteria: positive for EpCAM-APC (BioLegend catalog #118213) and negative for CD31-PE (eBioscience catalog #12-0311-81), CD45-PE (eBioscience catalog #12-0451-81), podoplanin-PE (eBioscience catalog #12-5381-80), Sca1-PE (eBioscience catalog #12-5981-81), CD24-PE (BioLegend catalog #119307), and DAPI (BioLegend catalog #422801) as previously described^{81 (link)}. To isolate mesenchymal cells we sorted for cells that were positive for CD140a (BioLegend catalog #135907) and negative for and DAPI (BioLegend catalog #422801). Antibody concentrations are in Supplementary Table 2.

Paris A.J., Hayer K.E., Oved J.H., Avgousti D.C., Toulmin S.A., Zepp J.A., Zacharias W.J., Katzen J.B., Basil M.C., Kremp M.M., Slamowitz A.R., Jayachandran S., Sivakumar A., Dai N., Wang P., Frank D.B., Eisenlohr L.C., Cantu E I.I.I., Beers M.F., Weitzman M.D., Morrisey E.E, & Worthen G.S. (2020). STAT3-BDNF-TrkB signaling promotes alveolar epithelial regeneration after lung injury. Nature cell biology, 22(10), 1197-1210.

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Hematopoietic Cell Phenotypic Analysis

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Hematopoietic cell phenotypic analysis was completed by flow cytometry. Briefly, 5 × 10⁶ hematopoietic cells harvested from mouse bone marrow were blocked with Fc-block for 10 min, incubated with biotin-conjugated lineage antibodies (CD4, CD8, CD45R/B220, Gr-1, Mac-1 and Ter-119) and then stained with streptavidin-FITC (BD Pharmingen), c-Kit-APC (BD Pharmingen), and Sca1-PE (eBioscience). The frequencies of HPCs (Lin-Sca1-c-kit1 + cells), LSK cells (Lin-Sca1 + c-kit + cells), and HSCs (Lin-Sca1 + c-kit + CD150 + CD48-cells) were analyzed with a flow cytometer (LSRII flow cytometer). Data were analyzed using FlowJo software.

Li C., Luo Y., Shao L., Meng A, & Zhou D. (2018). NOS2 deficiency has no influence on the radiosensitivity of the hematopoietic system. Cell & Bioscience, 8, 33.

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Cell Surface Marker Analysis by Flow Cytometry

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For cell surface flow cytometry, cells were incubated with human Fc receptor binding inhibitor (#14-9161−73; eBioscience) followed by primary antibodies CD235−APC (#306607; BioLegend) CD41-FITC (#303703; BioLegend) and CD11b-PE/Cy5 (#101209; BioLegend) or CD-71-APC (BD551374; BD Biosciences), p53-PECy7 (NB200-171; Novus), Sca-1-PE (#12-5981-81; eBioscience), CD34-Fluor®450 (#48-0341-82; eBioscience) and CD16/32-FITC (#101305; BioLegend). FRET measurements were obtained as described^{69 (link)}. To measure ECFP and FRET cells were excited with the 405 nm laser and fluorescence was collected in the ECFP channel with a standard 450/40 filter, while the FRET-signal was measured with a 529/24 filter. To measure EYFP, cells were excited with the 488 nm laser while emission was also taken with a 529/24 filter. Data were collected on a DxP10 flow cytometer (Cytek) and analyzed by using FlowJo Software, v.9.7.2.

Wilkes M.C., Siva K., Chen J., Varetti G., Youn M.Y., Chae H., Ek F., Olsson R., Lundbäck T., Dever D.P., Nishimura T., Narla A., Glader B., Nakauchi H., Porteus M.H., Repellin C.E., Gazda H.T., Lin S., Serrano M., Flygare J, & Sakamoto K.M. (2020). Diamond Blackfan anemia is mediated by hyperactive Nemo-like kinase. Nature Communications, 11, 3344.

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Single-Cell RNA-seq of Murine Mesenchymal Stem Cells

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For the initial scRNA-seq experiment was performed using the Chromium Single Cell Gene Expression Solution (10× Genomics), following the manufacturer’s protocol. The MSC was isolated from ten 6-week-old mice (all males). Cells were stained with the anti-mouse antibodies CD31 PE-Cy7, CD45 PE-Cy7 and TER119 PE-Cy7 (Biolegend), Sca-1 PE (eBioscience) and PDGFR-α APC (eBioscience), and 30,000 Lin − Sca-1 + Pdgfr-α + were isolated using a BD Bioscience FACS Aria III Fusion. Cells were washed and resuspended in 250 μl FACS buffer (PBS, 2% FBS, 1 mM EDTA), targeting the required 1000 cells/μl concentration, accounting for a 10–20% loss. We pipetted 9.7 μl cell suspension (concentration of 913 cells/μl, ~ 8800 cells), targeting the recovery of ~ 5000 cells. Single-cell RNA-seq libraries were obtained following the 10× Genomics recommended protocol, using the reagents included in the Chromium Single Cell 3’ v2 Reagent Kit. Libraries were sequenced on the NextSeq 500 v2 (Illumina) instrument using 150 cycles (18 bp barcode + UMI, and 132-bp transcript 3’ end), obtaining ~ 5 × 10⁸ raw reads^{46 (link),47 (link)}.

Kanazawa S., Okada H., Hojo H., Ohba S., Iwata J., Komura M., Hikita A, & Hoshi K. (2021). Mesenchymal stromal cells in the bone marrow niche consist of multi-populations with distinct transcriptional and epigenetic properties. Scientific Reports, 11, 15811.

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Multiparameter Analysis of BM Cell Subsets

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BM cells prepared above were stained with lineage markers on FITC (CD4, CD5, CD8, CD11b, B220, Gr1, Ter119, and CD41, eBioscience, San Diego, CA, USA), CD117-APC-H7 (BD Bioscience, San Jose, CA, USA), Sca1-PE (eBioscience), CD16/32-PerCP5.5 (BD Bioscience), CD150-PE-Cy7 (BioLegend, San Diego, CA, USA), and CD105-BV421 (BD Bioscience) for 30 min on ice and gating as described by Pronk et al (34 (link)). and Singer et al. (12 (link)). Dead cells were excluded using 7-Amino-Actinomycin D (7-AAD; eBioscience) and AccuCheck counting beads (PCB100, Invitrogen) were used for quantification of absolute cell numbers. Multiparameter analysis was performed on a FACSSymphony A3 (BD Bioscience) using the FACSDiva software and data were analyzed using the FlowJo software (version 10, Tree Star Inc., Ashland, OR, USA).

Kim Y., Lee Y., Lee M.N., Nah J., Yun N., Wu D, & Pae M. (2022). Time-restricted feeding reduces monocyte production by controlling hematopoietic stem and progenitor cells in the bone marrow during obesity. Frontiers in Immunology, 13, 1054875.

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Mesenchymal Stem Cell Characterization

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The standard reagents and chemicals were obtained from Sigma (St. Louis, MO). Recombinant cytokines and antibodies of IL-6, IL-12, TNF-α, and IL-10 were purchased from BD Biosciences (San Diego, CA). Fluorochrome-labelled antibodies (CD29 FITC, CD34 eFluor 660, CD44 PerCP-Cyanine5.5, CD45 APC, and Sca-1 PE) were purchased from eBiosciences (San Diego, CA) unless otherwise mentioned. Fetal bovine serum (FBS) was from GIBCO. LPS and N-glycolyl MDP used as ligands for TLR-4 and NOD-2 in the experiments were procured from InvivoGen (San Diego, CA). Oil Red-O stain was bought from Himedia (Mumbai, India). Alizarin Red S stain was acquired from Sigma (St. Louis, MO).

Aqdas M., Singh S., Amir M., Maurya S.K., Pahari S, & Agrewala J.N. (2021). Cumulative Signaling Through NOD-2 and TLR-4 Eliminates the Mycobacterium Tuberculosis Concealed Inside the Mesenchymal Stem Cells. Frontiers in Cellular and Infection Microbiology, 11, 669168.

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Leukemia Stem Cell Immunophenotyping in Mice

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For leukemia stem cell analysis, bone marrow cells were collected from primary BMT recipient mice and resuspended in ammonium chloride solution to lysate red cells. After washing, cells were stained at 4°C with various antibodies diluted in Flow Cytometry Staining Buffer (eBioscience) for 30 minutes. After incubation, cells were resuspended in IC Fixation Buffer (eBioscience) before being loaded for flow cytometry analysis in BD FACS FortessaX-20. The following antibodies were used for flow cytometry: anti-CD45.2-FITC (109806, BioLegend), anti-lineage markers-eFluor^™ 450 (88-7772-72, eBioscience), c-Kit-APC (17-1171-82, eBioscience), Sca-1-PE (12-5981-82, eBioscience), CD16/CD32-APC/Cyanine7 (156612, BioLegend), and CD34-PE/Cyanine7 (119326, BioLegend).

Han L., Dong L., Leung K., Zhao Z., Li Y., Gao L., Chen Z., Xue J., Qing Y., Li W., Pokharel S.P., Gao M., Meiling C.h.e.n., Shen C., Tan B., Small A., Wang K., Zhang Z., Qin X., Yang L., Wunderlich M., Zhang B., Mulloy J.C., Marcucci G., Chen C.W., Wei M., Su R., Chen J, & Deng X. (2023). METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism. Cell stem cell, 30(1), 52-68.e13.

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Multiparameter Flow Cytometry Analysis of Hematopoietic Stem and Progenitor Cells

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Cells were stained and analyzed on either a FACSCalibur II or LSR II (Becton Dickinson, San Jose, CA). The following anti-mouse antibodies were used for flow cytometry analysis: B220-PE, CD3e-FITC, CD11b-APC, Gr-1-APC-Cy7, CD48-Pacific Blue, CD150-PE-Cy7, CD34, c-Kit-APC, Sca-1-PE, FcγR-PE, CD45.1-Brilliant Violet 570, and CD45.2-Alexa Fluor 700 (all from eBiosciences, San Diego, CA). Cell sorting was performed using a FACS Aria (Becton Dickinson, San Jose, CA). Antibodies for Annexin V and Ki-67 assays were used as per manufacturer’s protocol (Becton Dickinson, San Jose, CA). Gating strategy for HSPC fractions are as follows: (1) LSK: Lineage^NegCkit+Sca1+; (2) CMP: Lineage^NegCkit+Sca1^Neg CD34+FcγRIII^low; (3) GMP: Lineage^NegCkit+Sca1^Neg CD34+FcγRIII^high; (4) MEP: Lineage^NegCkit+Sca1^Neg CD34^NegFcγRIII^Neg; (5) CLP: Lineage^Neg IL7Rα^highCkit+Sca1^low; ProB Frac A: B220^lowCD43^HighBP1^NegHSA^Neg; ProB Frac B: B220^lowCD43^HighBP1^NegHSA^Pos; ProB Frac C: B220^lowCD43^HighBP1^HighHSA^Pos; ProB Frac D: B220^HighCD43^NegIgM^Neg; ProB Frac E: B220^HighCD43^NegIgM^Mid; ProB Frac F: B220^High++CD43^NegIgM^High/Mid.

Wheat J.C., Krause D.S., Shin T.H., Chen X., Wang J., Ding D., Yamin R, & Sweetser D.A. (2014). The Corepressor Tle4 Is a Novel Regulator of Murine Hematopoiesis and Bone Development. PLoS ONE, 9(8), e105557.

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