Cd34 percp cy5

Manufactured by BioLegend

Sourced in United States

CD34-PerCP-Cy5.5 is a fluorescently-labeled antibody used to detect the CD34 antigen, a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells. The antibody is conjugated with the PerCP-Cy5.5 fluorescent dye, which can be detected using flow cytometry equipment.

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Lab products found in correlation

Cd45 apc cy7, by BioLegend (3 mentions) Cd14 apch7 m p9, by BD (2 mentions) Cmpo fitc sc 51741 fitc, by Santa Cruz Biotechnology (2 mentions) γh2ax alexa488, by BD (2 mentions) Cd11b apc, by BioLegend (2 mentions) Fitc annexin 5 apoptosis detection kit 1, by BD (2 mentions) Cd73 pe cy7, by BioLegend (2 mentions) Cd31 pe, by BioLegend (2 mentions) Cd274 pe, by BioLegend (2 mentions) Cd105 pe, by BioLegend (2 mentions) Cd31 apc cy7, by BioLegend (1 mentions) Collagenase type 2, by Thermo Fisher Scientific (1 mentions) Cd90 alexa fluor 647, by BioLegend (1 mentions) Pdgfr, by BioLegend (1 mentions) Live dead fixable aqua, by BioLegend (1 mentions) Anti cd90 apc, by BioLegend (1 mentions) Facscanto 2 cytometer, by BD (1 mentions) Gm csf, by Thermo Fisher Scientific (1 mentions) Cd14 alexa fluor 700, by BioLegend (1 mentions) Cd11b pe cy7, by BioLegend (1 mentions)

8 protocols using cd34 percp cy5

Isolation and Characterization of Human Adipose Cells

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Adipose tissue was digested with Type II collagenase (175 units/ml PBS/2% BSA, Life Technologies Inc., Carlsbad, CA, USA) 60 minutes, 37°C, centrifuged, and the SVF cell pellet used for RNA or flow cytometry. Mature adipocytes were harvested from the adipocyte layer of collagenase-digested tissue followed by RNA isolation. Preadipocytes were isolated as described^{25 (link)}: SVF were plated overnight, non-adherent cells discarded, and adherent cells passaged three times to enrich for preadipocytes followed by RNA isolation. Such cells demonstrate adipocyte differentiation capacity and retain depot- and patient-specific characteristics^{26 (link)}.
For flow cytometry, SVF cells were stained with viable dye and antibodies and analyzed on a FACSCanto II flow cytometer (Becton-Dickinson Inc., Franklin Lakes, NJ, USA). Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA) after exclusion of doublets and non-viable cells using fluorescence-minus-one controls with forward scatter/side scatter gates encompassing all cells with subsequent analysis of CD45- cells. Preadipocytes were defined as CD45-CD34+CD31-. Antibodies: CD45-FITC, CD31-APC-Cy7, CD34-PERCP-Cy5.5 (Biolegend Inc., San Diego, CA, USA), anti-rabbit secondary antibody-PE (Life Technologies, Inc., Carlsbad, CA, USA); CD140a-AlexaFluor 647 (BD Biosciences, Inc., San Jose, CA, USA).

Muir L.A., Neeley C.K., Meyer K.A., Baker N.A., Brosius A.M., Washabaugh A.R., Varban O.A., Finks J.F., Zamarron B.F., Flesher C.G., Chang J.S., DelProposto J.B., Geletka L., Martinez-Santibanez G., Kaciroti N., Lumeng C.N, & O'Rourke R.W. (2016). Adipose tissue fibrosis, hypertrophy, and hyperplasia: correlations with diabetes in human obesity. Obesity (Silver Spring, Md.), 24(3), 597-605.

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Apoptosis and Cell Differentiation Assay

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Apoptosis staining was conducted using the FITC Annexin V Apoptosis Detection Kit I (BD Bioscience, San Jose, CA, USA). Staining for differentiation markers was performed using the following antibodies directed against cell surface markers: CD34-PerCPCy5.5 (#343611; Biolegend, San Diego, CA, USA), CD11b-APC (#301309; BioLegend, San Diego, CA, USA), CD14-APCH7 (MϕP9; BD Biosciences, San Jose, CA, USA). For intracellular staining, either one of the following antibodies were used: cMPO-FITC (sc-51741 FITC; Santa Cruz Biotechnology, Dallas, TX, USA) or γH2AX-Alexa488 (560445, BD Biosciences, San Jose, CA, USA). A detailed staining protocol is attached in the supplemental methods.

Kiehlmeier S., Rafiee M.R., Bakr A., Mika J., Kruse S., Müller J., Schweiggert S., Herrmann C., Sigismondo G., Schmezer P., Krijgsveld J, & Gröschel S. (2021). Identification of therapeutic targets of the hijacked super-enhancer complex in EVI1-rearranged leukemia. Leukemia, 35(11), 3127-3138.

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Apoptosis and Differentiation Analysis

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Apoptosis staining was conducted using the FITC Annexin V Apoptosis Detection Kit I (BD Bioscience, San Jose, CA, USA). Staining for differentiation markers was performed using the following antibodies directed against cell surface markers: CD34−PerCPCy5.5 (#343611; Biolegend, San Diego, CA, USA), CD11b−APC (#301309; BioLegend, San Diego, CA, USA), CD14−APCH7 (MϕP9; BD Biosciences, San Jose, CA, USA). For intracellular staining, either one of the following antibodies were used: cMPO-FITC (sc-51741 FITC; Santa Cruz Biotechnology, Dallas, TX, USA) or γH2AX-Alexa488 (560445, BD Biosciences, San Jose, CA, USA). A detailed staining protocol is attached in the Supplementary methods.

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Fibroblast Immunophenotyping Workflow

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Cultured fibroblasts (passages 0-2) were grown in culture to 80% to 90% confluence and trypsinized, as previously described.^{4 (link)} Single-cell suspensions were aliquoted to a 96-well plate. Cells were stained with the following antibody panel: LIVE/DEAD Fixable Aqua, CD31 (PECAM) Brilliant Violet 421 (303124; BioLegend), CD34 PerCP-CY5.5 (343612; BioLegend), FAPa PE (FAB3715P-025; R&D Systems), CD90 Alexa Fluor 647 (328120; BioLegend), and PDGFR (platelet-derived growth factor receptor) PE/Cy7 (323508; BioLegend). After staining, cells were fixed and permeabilized. Single-stained and florescence minus one–stained samples were used as controls for gate selection. Viability Aqua–negative (live) cells were subsequently evaluated by percentage population of fibroblasts (FAP⁺). Fibroblast cells line were grouped according to etiology (n = 3), with rapidly processed autopsy tracheal mucosa–derived nonscar fibroblasts used as controls. All analyses were performed in FlowJo Flow Cytometry Analysis Software (Treestar, Version 10.7.1 for Mac).

Lina I.A., Berges A., Ospino R., Davis R.J., Motz K.M., Tsai H.W., Collins S, & Hillel A.T. (2021). Identifying Phenotypically Distinct Fibroblast Subsets in Type 2 Diabetes–Associated Iatrogenic Laryngotracheal Stenosis. Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery, 166(4), 712-719.

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Comprehensive Immunophenotypic Characterization

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hWJ-MSCs were immunophenotypically characterized by labeling the cells with anti-CD90-APC, -CD73-PE/Cy7, -CD105-PE, -CD274-PE, -CD45-APC/Cy7, -CD34-PerCP- Cy5.5, and -CD31-PE antibodies (Biolegend, San Diego, USA). T-cell depletion lymphocytes (TCD3⁺) inhibition was evaluated and cell suspensions were analyzed for sterility, endotoxins, and mycoplasma. A FACSCanto II cytometer (BD, Franklin Lakes, NJ, USA) and FlowJo vX.7.0 software (TreeStar, USA) was used for flow cytometry and data analysis, respectively.

Evangelho K.D., Cifuentes-González C., Rojas-Carabali W., Vivero-Arciniegas C.D., Cañas-Arboleda M., Salguero G., Ramírez-Santana C, & de-la-Torre A. (2024). Early detection of optic nerve head changes using optical coherence tomography after using mesenchymal stromal cells as intravitreal therapy in rabbit models of ocular hypertension. Veterinary World, 17(2), 500-508.

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Myeloid Lineage Differentiation from CD34+ Cells

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Myeloid lineage-specific CD34⁺ differentiation was carried out by culturing the cells in expansion media supplemented with 20 ng/mL GM-CSF (Peprotech, ref. 300-03). Cells were cultured at 1 × 10⁵–1 × 10⁶ cells/mL densities and analyzed on day 7 and day 14, using CD34⁺-PerCP-Cy5.5 (1:60, clone 561), CD11b-PE-Cy7 (1:60, clone ICRF44), CD14-AlexaFluor700 (1:200, clone 63D3), CD16-APC (1:40, clone B73.1), and CD33-PE conjugated antibodies (1:60, clone P67.6) (all from BioLegend). Myeloid differentiation of AML cell lines was assessed using CD11b-PE-Cy7 (1:60, Biolegend clone ICRF44).

Piragyte I., Clapes T., Polyzou A., Klein Geltink R.I., Lefkopoulos S., Yin N., Cauchy P., Curtis J.D., Klaeylé L., Langa X., Beckmann C.C., Wlodarski M.W., Müller P., Van Essen D., Rambold A., Kapp F.G., Mione M., Buescher J.M., Pearce E.L., Polyzos A, & Trompouki E. (2018). A metabolic interplay coordinated by HLX regulates myeloid differentiation and AML through partly overlapping pathways. Nature Communications, 9, 3090.

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Immunophenotypic Characterization of Expanded WJ-MSCs

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Expanded WJ-MSC (n = 3) supplemented with 10% hPL (n = 3, batch 50, 53, 54), hS (n = 3, batch 21, 54, 57) and FBS (n = 2, batch 1, 2) (Gibco, Life Technologies, Carlsbad, CA, USA) were characterized immunophenotypically using antibodies against the following human antigens—CD90-APC, CD73-PE/Cy7, CD105-PE, CD274-PE, CD45-APC/Cy7, CD34-PerCP-Cy5.5, CD31-PE and HLA-DR-Pacific Blue (Biolegend, San Diego, USA). Appropriate isotype controls for each of the antibodies were used. Cells cultures a 70–80% confluence were harvested using 0.25% trypsin-EDTA, (Gibco, Life Technologies, Carlsbad, CA, USA) and centrifuged at 1200 rpm × 6 min. Cell pellets were resuspended in 100 µL of 1× PBX with 5% Bovine Serum Albumin (Gibco, Life Technologies, Carlsbad, CA, USA) containing the corresponding antibody. Cell suspension was incubated for 30 min at 4 °C, washed with 1X PBS and resuspended in 200 µL 1× PBS. Flow cytometry analysis were carried out with a FACSCanto II™ instrument (BD, Franklin Lakes, NJ, USA) and data were analyzed with the FlowJo vX.7.0 software package (TreeStar, USA).

Cañas-Arboleda M., Beltrán K., Medina C., Camacho B, & Salguero G. (2020). Human Platelet Lysate Supports Efficient Expansion and Stability of Wharton’s Jelly Mesenchymal Stromal Cells via Active Uptake and Release of Soluble Regenerative Factors. International Journal of Molecular Sciences, 21(17), 6284.

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Characterizing ccRCC Tumor Cell Subsets

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Six ccRCC specimens were obtained from the University Health Network (UHN) Program in Biospecimen Sciences from consenting patients in accordance with the policies of the UHN Research Ethics Board (REB ID# 09–0828). Flow cytometry was performed as previously described [27 (link)]. Briefly, primary ccRCC specimens were disaggregated into single cell suspensions using collagenase treatment. The specimens were stained using CD45-APC-Cy7 (1:200), CD31-PE-Cy7 (1:200), CD34-PerCP-Cy5.5 (1:50) (all BioLegend) and 1.2 mg/ml TE7-biotin (in-house production from hybridoma obtained from ATCC) followed by Streptavidin-eFluor450 (1:400; eBioscience). These cells were then stained as described in Gedye et al. with an antibody panel that included other stem/progenitor and immune markers [27 (link)]. Data collection was performed on a Becton-Dickinson LSR II flow cytometer.

Yoon J.Y., Gedye C., Paterson J, & Ailles L. (2020). Stem/progenitor cell marker expression in clear cell renal cell carcinoma: a potential relationship with the immune microenvironment to be explored. BMC Cancer, 20, 272.

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