iPSCs (DF19-19-9-11T) purchased from WiCell Technologies were maintained on vitronectin-coated plates in Essential 8 medium (ThermoFisher Scientific) and passaged when the colonies reached ~75-80% confluence. iPSCs were differentiated into CD34+ iPSC-EPs following an established low-serum medium protocol [39 (link)]. Five days after CHIR induction (LC Technologies), cells were purified by methods we have described previously [35 (link)]. Briefly, the resulting confluent monolayer of differentiated cells was dispersed into a single-cell suspension with Accutase, tagged with a CD34-PE antibody (Miltenyi Biotec), and purified on a fluorescence-activated cell sorting (FACS) instrument (S3e Cell Sorter, Bio-Rad). After sorting, CD34+ iPSC-EPs were encapsulated at 2.5 million cells/mL in collagen-only or IPN hydrogels, as described in Section 2.4 (Table S1 ). After encapsulation, cells were cultured in EGM-2 (PromoCell) supplemented with 10 uM Y-27632, 1X Penicillin-Streptomycin, and 50 ng/mL vascular endothelial growth factor (VEGF) for 24 hours. The medium was then refreshed daily with EGM-2 supplemented with 50 ng/mL VEGF.
Cd34 pe antibody
Manufactured by Miltenyi Biotec
The CD34-PE antibody is a fluorescent-labeled antibody that specifically binds to the CD34 antigen, which is expressed on the surface of hematopoietic stem and progenitor cells. This antibody can be used for the identification and isolation of CD34-positive cells in flow cytometry and cell sorting applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Essential 8 medium, by Thermo Fisher Scientific (1 mentions)
Accutase, by Miltenyi Biotec (1 mentions)
S3e cell sorter, by Bio-Rad (1 mentions)
Egm 2, by PromoCell (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Inside stain kit, by Miltenyi Biotec (1 mentions)
Zen 2011, by Zeiss (1 mentions)
2 protocols using cd34 pe antibody
1
iPSC-derived Endothelial Progenitor Cell Encapsulation
2
Rab5b Localization in Cells using Confocal Microscopy
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For siRNA localisation studies, cell surface was stained with CD34-PE antibody (Miltenyi Biotec, clone: AC136). After washing with PBS, cells were fixed and permeabilised in suspension using the Inside stain Kit (Miltenyi Biotec) mainly following the manufacturer’s instruction. Inside fix was used without dilution and cells were pelleted by centrifugation for 5 min at 600 × g. All labelling steps were performed at RT. Rabbit-anti-Rab5b antibody staining (Santa Cruz, sc-598) was performed using a 1:50 dilution for 30 min following incubation with anti-rabbit-IgG-AF594 antibody (Life technologies, A-11012) in a 1:100 dilution for 30 min. For nuclear counterstaining, DRAQ5 (ebioscience) was added at a final concentration of 1.5 μM and incubated for 10 min. Cells were seeded into 8 well CytoCapture chambers (Miltenyi Biotec, H20-10) and analysed with a LSM710 confocal microscope (Zeiss) using a 63× immersion objective. Pictures were processed with the Carl Zeiss ZEN 2011 software.
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