Cd34 alexafluor647

Manufactured by BioLegend

CD34-AlexaFluor647 is a fluorescently-labeled monoclonal antibody that binds to the CD34 cell surface antigen. CD34 is a glycoprotein expressed on hematopoietic stem and progenitor cells. The Alexa Fluor 647 dye provides a bright, photostable fluorescent signal for detection and analysis.

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Lab products found in correlation

Cd29 alexafluor700, by BioLegend (3 mentions) Cd31 pe cy7, by Thermo Fisher Scientific (2 mentions) Ps473 akt, by Cell Signaling Technology (2 mentions) Pan akt, by Cell Signaling Technology (2 mentions) Pt308 akt, by Cell Signaling Technology (2 mentions) Cd45 fitc, by Thermo Fisher Scientific (2 mentions) Antibodies to irβ, by Cell Signaling Technology (2 mentions) Live dead blue, by Thermo Fisher Scientific (2 mentions) Sca1 pacific blue, by BD (2 mentions) Cl316 243, by Bio-Techne (2 mentions) Cd45 pe cy7, by Thermo Fisher Scientific (2 mentions) Cd11b pb, by BioLegend (1 mentions) Cd45 pacific blue, by BioLegend (1 mentions) Pe cy7 cd105, by BioLegend (1 mentions) Cd45 pe cy7, by BioLegend (1 mentions) Fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions) Lsrfortessa analyzer, by BD (1 mentions) Cd31 pe cy7, by BioLegend (1 mentions) Anti ki67 fitc antibody, by Thermo Fisher Scientific (1 mentions) Cd140a pe, by BioLegend (1 mentions)

5 protocols using cd34 alexafluor647

Antibody-based Adipose Tissue Characterization

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Antibodies to IRβ, P-T308-Akt, P-S473-Akt and Pan-Akt (all used at 1/1000 dilution) were from Cell Signaling. UCP1 antibody (#10983) was from AbCam (used at 1/100 for IHC and at 1/1000 for western blot). Live/dead Blue (Molecular Probes, L23105), CD31-PE-CY7 (eBioscience, 25-0311)(used at 1/1000), CD45-PE-CY7 (eBioscience, 25-0451) (used at 1/1000), CD29-AlexaFluor700 (Biolegend, 102218)(used at 1/400), CD34-AlexaFluor647 (Biolegend, 119314)(used at 1/200), Sca1-Pacific Blue (BD Bioscience, 560653)(used at 1/1000), CD45-FITC (eBioscience, 11-0451) (used at 1/200) were used for flow cytometry. CL316,243 was from Tocris.

Sanchez-Gurmaches J, & Guertin D.A. (2014). Adipocytes arise from multiple lineages that are heterogeneously and dynamically distributed. Nature communications, 5, 4099.

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Phenotypic Characterization of ASC

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ASC cultures from early passages (1–6 population doublings) were harvested and resuspended in DMEM/F12 with 2% FBS. The cells were then centrifuged and suspended in cold PBS at a concentration of 10⁶ cells/100 μl. Cell aliquots were stained with monoclonal mouse anti-human antibodies against the following antigens: CD44-peridinin-chlorophyll-protein/cyanin 5.5, CD90-phycoerthyrin (PE), PE/Cy7-CD105, CD73-fluorescein isothiocyanate, CD34-Alexa Fluor 647, CD45-Pacific Blue (PB), CD11b-PB, and CD31-PB (BioLegend, San Diego, CA) for 30 minutes in the absence of light at 4°C. The cells that were stained with a single antibody coupled with a fluorescent dye were acquired for compensation purposes. After incubation, the cells were washed and resuspended in cold PBS. Flow cytometry was performed using a Becton Dickinson LSR II. A gate was set to include only the viable propidium iodide-negative cells. The number of cells staining positive for a given cell surface marker was determined by the percentage of cells present within an established gate. A minimum of 10,000 events were counted for each analysis.

Hariharan N., Ashcraft K.A., Svatek R.S., Livi C.B., Wilson D., Kaushik D., Leach R.J, & Johnson-Pais T.L. (2018). Adipose Tissue-Secreted Factors Alter Bladder Cancer Cell Migration. Journal of Obesity, 2018, 9247864.

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Antibody-based Adipose Tissue Characterization

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Sanchez-Gurmaches J, & Guertin D.A. (2014). Adipocytes arise from multiple lineages that are heterogeneously and dynamically distributed. Nature communications, 5, 4099.

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Adipose Tissue Stromal Cell Isolation

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Isolation of adipose tissue stromal cells was performed essentially as described20 (link). Briefly, adipose tissue was excised, minced, and digested in Hank’s Balanced Salt Solution (HBSS) (Sigma no. H8264) containing 3% BSA and 0.8 mg/ml Collagenase Type 2 (Worthington Biochemical; LS004174) for 75 min in a shaking water bath at 37 °C. The mixture was then filtered through a 40 μm filter, and filtered cells were pelleted and washed in HBSS containing 3% BSA. For Ki67 analysis, cells were stained with the following antibodies at room temperature: CD45 PE-Cy7 (Biolegend; clone 30-F11), CD31 PE-Cy7 (Biolegends; clone 390), CD29 Alexa Fluor 700 (BioLegend; clone HMβ1-1), CD34 Alexa Fluor 647 (BioLegend; clone MEC14.7) and CD140a PE (Biolegend; clone APA5). Anti-Ki67 FITC antibody (eBioscience, clone SOLA15) was used overnight at 4 °C. Cells
were washed, fixed and permeabilized using the Fixation/Permeabilization kit (eBioscience) as per manufacturer’s protocol. All antibody incubations and washing steps were performed in HBSS with 3% BSA. Following antibody incubation, samples were analyzed on a BD LSR Fortessa analyzer. Data analysis was performed using BD FACS Diva software (BD Biosciences).

Wagner G., Lindroos-Christensen J., Einwallner E., Husa J., Zapf T.C., Lipp K., Rauscher S., Gröger M., Spittler A., Loewe R., Gruber F., Duvigneau J.C., Mohr T., Sutterlüty-Fall H., Klinglmüller F., Prager G., Huppertz B., Yun J., Wagner O., Esterbauer H, & Bilban M. (2017). HO-1 inhibits preadipocyte proliferation and differentiation at the onset of obesity via ROS dependent activation of Akt2. Scientific Reports, 7, 40881.

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FACS Sorting of CD34+ Cells

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For FACS sorting of primary cells, mononuclear PB or BM cells were suspended in Hanks' Balanced Salt Solution (HBSS, STEMCELL Technologies) with 2% FBS and stained for 30 min at 4 C with anti-human CD38-Phycoerythrin (PE, 1:50, BD Biosciences) or antihuman CD38-Brilliant Violett 711 (BV711, 1:50, BioLegend) and CD34-APC (1:100, Thermo Fisher Scientific) or CD34-Alexa Fluor 647 (1:100, BioLegend) antibodies. CD34-subpopulations were sorted on a FACS AriaFusion (BD).

Rothe K., Babaian A., Nakamichi N., Chen M., Chafe S.C., Watanabe A., Forrest D.L., Mager D.L., Eaves C.J., Dedhar S, & Jiang X. (2020). Integrin-Linked Kinase Mediates Therapeutic Resistance of Quiescent CML Stem Cells to Tyrosine Kinase Inhibitors. Cell stem cell, 27(1).

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