iECs were derived using a three-dimensional approach with modifications 10 (link). Briefly, to initiate differentiation, iPSCs were cultured in ultra-low, non-adhesive dishes to form embryoid body (EB) aggregates in EBM2 media (Lonza) in the absence of leukemia inhibitor factor (LIF). After 4 days of suspension culture, the EBs were reattached onto 0.2% gelatin-coated dishes and cultured in EBM2 medium supplemented with VEGF-A165 (50 ng/mL; PeproTech). After 3 weeks of differentiation, single cell suspensions were obtained using a cell dissociation buffer (Life Technologies) and labeled with APC-conjugated CD31 (eBiosciences) and PE-conjugated CD144 (BD Biosciences) anti-mouse antibodies. iECs were purified by fluorescence activated cell sorting (FACS) of CD31+CD144+ population. iECs were maintained in EBM2 media supplemented with recombinant murine vascular endothelial growth factor (50 ng/ml).
Apc conjugated cd31
Manufactured by Thermo Fisher Scientific
APC-conjugated CD31 is a reagent used in flow cytometry applications. CD31, also known as PECAM-1, is a cell surface marker expressed on endothelial cells, platelets, and certain subsets of leukocytes. The APC (Allophycocyanin) fluorescent dye is conjugated to the CD31 antibody, allowing for the detection and analysis of CD31-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Cell dissociation buffer, by Thermo Fisher Scientific (2 mentions)
Pe conjugated cd144, by BD (2 mentions)
Vegf a165, by Thermo Fisher Scientific (2 mentions)
Ebm 2 media, by Lonza (2 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Pe conjugated cd105, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated cd29, by Thermo Fisher Scientific (1 mentions)
Percp cy5.5 conjugated cd45, by Thermo Fisher Scientific (1 mentions)
Hyaluronidase type 4 s, by Merck Group (1 mentions)
Pe conjugated cd 133, by Thermo Fisher Scientific (1 mentions)
Dnase 1 grade 2, by Roche (1 mentions)
Liberase tm research grade, by Roche (1 mentions)
70 μm nylon cell strainer, by BD (1 mentions)
Dispase, by Roche (1 mentions)
4 protocols using apc conjugated cd31
1
Derivation of Induced Endothelial Cells
2
Multiparameter Flow Cytometry Panel
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Antibodies were purchased from the following sources: PE conjugated CD105, FITC conjugated CD29, APC conjugated CD31, PerCP-Cy5.5 conjugated CD45, and PE-Cy7 conjugated CD34 were from eBioscience. p63, AR, CK8, CK5, PSA and vimentin antibodies were from Santa Cruz Biotechnology. Testosterone and collagenase IV were from Sigma and human nuclei antibody was from Millipore.
3
Derivation of Induced Endothelial Cells
4
Flow Cytometric Analysis of Angiogenic Cell Types
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Subcutaneous matrigel plugs were digested for 60–90 minutes at 37 °C in an enzymatic cocktail composed of 25 μg/ml DNase-I, grade II (Roche, cat no. 10-104-159-001), 3 U/ml Dispase (Roche, cat no. 10-269-638-001), 3 U/ml Liberase TM Research grade (Roche, cat no. 05-401-119-001) and 25 μg/ml Hyaluronidase Type IV-S (Sigma, cat no. H3884). The isolated cells were filtered through a 70 μm Nylon cell strainer (BD Falcon, Bedford, MA) into a sterile 50 ml centrifuge tube and cells washed 3X with FACS Buffer (1X PBS without Ca/Mg, 2 mM EDTA and 0.5% BSA).
Isolated cells were analyzed by flow cytometry to determine their cellular characteristics. FITC-conjugated F4/80 (eBioscience, cat no.11-4801) and APC-conjugated CD31 (eBioscience, cat no. 17-0311) were used to distinguish endothelial cells from macrophages with additional labeling using eFluor450-conjugated CD34 (eBioscience, cat no. 48-0341), PE-conjugated CD-133 (eBioscience, cat no. 12-1331) or PERC-P conjugated CD45 (eBioscience, cat no. 45-0451). Antibodies were incubated with isolated cells in FACS buffer for 30 minutes at 4 °C in darkness. Cells were washed 3X with FACS buffer and fixed in 1% PFA in PBS for 10 minutes at room temperature in the absence of light. Fixed cells were washed in PBS and resuspended in 400 μl of FACS buffer for flow cytometry. Experiment performed three times.
Isolated cells were analyzed by flow cytometry to determine their cellular characteristics. FITC-conjugated F4/80 (eBioscience, cat no.11-4801) and APC-conjugated CD31 (eBioscience, cat no. 17-0311) were used to distinguish endothelial cells from macrophages with additional labeling using eFluor450-conjugated CD34 (eBioscience, cat no. 48-0341), PE-conjugated CD-133 (eBioscience, cat no. 12-1331) or PERC-P conjugated CD45 (eBioscience, cat no. 45-0451). Antibodies were incubated with isolated cells in FACS buffer for 30 minutes at 4 °C in darkness. Cells were washed 3X with FACS buffer and fixed in 1% PFA in PBS for 10 minutes at room temperature in the absence of light. Fixed cells were washed in PBS and resuspended in 400 μl of FACS buffer for flow cytometry. Experiment performed three times.
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