For each sample, a calculated volume of bioreactor‐generated aggregates containing 1E6 cells per stain were enzymatically dissociated into a single‐cell suspension, using the dissociation method described above, and resuspended in fixation buffer (Cat#FC001, R&D Systems) to be incubated for 15 minutes at room temperature. Dissociated cells were rinsed twice with PBS then resuspended in 200 μL of permeabilization buffer (Cat#FC005, R&D Systems) with 1 μg/106 cells antibody stain and 1 μM/106 cells nuclei stain and incubated for 1 hour at room temperature. Conjugated antibody stains for TRA‐1‐60 (Ca # FAB4770P, R&D Systems) and Nanog (Cat#MABD24A4, Millipore Sigma) were used along with the nuclei stain To‐Pro‐3 Iodide Nucleic Acid Stain (Cat#T3605, Thermo Fisher). Cells were then rinsed twice with PBS and imaged using a Carl Zeiss Laser Scanning Microscope 700 with lasers at 488 and 639 nm and corresponding filter sets.
Fab4770p
Manufactured by R&D Systems
The FAB4770P is a lab equipment product manufactured by R&D Systems. It is a versatile piece of equipment designed for use in various laboratory settings. The core function of this product is to provide researchers with a reliable and efficient tool for their experimental needs.
Automatically generated - may contain errors
4 protocols using fab4770p
1
Quantifying Pluripotency Markers in Cells
2
Immunofluorescence Analysis of Stem Cells
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Aggregate samples containing 1E6 cells were removed from the bioreactor culture and added into microcentrifuge tubes (Cat#10011-724, VWR). Aggregates were rinsed twice with PBS and resuspended in 0.5 mL of fixation buffer (Cat#FC001, R&D Systems) to be incubated for 1 h at room temperature. Aggregate samples were then rinsed twice with PBS and resuspended in 200-μL permeabilization buffer (Cat#FC005, R&D Systems) with 1 μg/106 cells antibody stain and 1 μM/106 cells nuclei stain and incubated for 3 h at room temperature. Conjugated antibody stains for SSEA-4 (Cat#FAB1435F, R&D Systems), TRA-1-60 (Cat# FAB4770P, R&D Systems), and Nanog (Cat#MABD24A4, Millipore Sigma) were used along with the nuclei stain To-Pro-3 Iodide Nucleic Acid Stain (Cat#T3605, Thermo Fisher). Cells were then rinsed twice with PBS and imaged using a Carl Zeiss Laser Scanning Microscope 700 with lasers at 488 nm and 639 nm and corresponding filter sets.
3
Pluripotent Stem Cell Characterization
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Aggregate samples containing 1E6 cells were removed from the bioreactor culture and added into microcentrifuge tubes (Cat#10011-724, VWR). Aggregates were rinsed twice with PBS and resuspended in 0.5 mL of fixation buffer (Cat#FC001, R&D Systems) to be incubated for 1 hr at room temperature. Aggregate samples where then rinsed twice with PBS and resuspended in 200 L permeabilization buffer (Cat#FC005, R&D Systems) with 1 g/10 6 cells antibody stain and 1 M/10 6 cells nuclei stain and incubated for 3 hrs at room temperature. Conjugated antibody stains for SSEA-4 (Cat#FAB1435F, R&D Systems), TRA-1-60 (Cat# FAB4770P, R&D Systems)
and Nanog (Cat#MABD24A4, Millipore Sigma) were used along with the nuclei stain To-Pro-3
Iodide Nucleic Acid Stain (Cat#T3605, Thermo Fisher). Cells were then rinsed twice with PBS and imaged using a Carl Zeiss Laser Scanning Microscope 700 with lasers at 488 nm and 639 nm and corresponding filter sets.
and Nanog (Cat#MABD24A4, Millipore Sigma) were used along with the nuclei stain To-Pro-3
Iodide Nucleic Acid Stain (Cat#T3605, Thermo Fisher). Cells were then rinsed twice with PBS and imaged using a Carl Zeiss Laser Scanning Microscope 700 with lasers at 488 nm and 639 nm and corresponding filter sets.
4
Immunophenotyping of Stem Cell Aggregates
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Aggregate samples containing 1E6 cells were removed from the bioreactor culture and added into microcentrifuge tubes (Cat#10011-724, VWR). Aggregates were rinsed twice with PBS and resuspended in 0.5 mL of fixation buffer (Cat#FC001, R&D Systems) to be incubated for 1 hr at room temperature. Aggregate samples where then rinsed twice with PBS and resuspended in 200 L permeabilization buffer (Cat#FC005, R&D Systems) with 1 g/10 6 cells antibody stain and 1 M/10 6 cells nuclei stain and incubated for 3 hrs at room temperature.
Conjugated antibody stains for SSEA-4 (Cat#FAB1435F, R&D Systems), TRA-1-60 (Cat# FAB4770P, R&D Systems) and Nanog (Cat#MABD24A4, Millipore Sigma) were used along with the nuclei stain To-Pro-3 Iodide Nucleic Acid Stain (Cat#T3605, Thermo Fisher). Cells were then rinsed twice with PBS and imaged using a Carl Zeiss Laser Scanning Microscope 700 with lasers at 488 nm and 639 nm and corresponding filter sets.
Conjugated antibody stains for SSEA-4 (Cat#FAB1435F, R&D Systems), TRA-1-60 (Cat# FAB4770P, R&D Systems) and Nanog (Cat#MABD24A4, Millipore Sigma) were used along with the nuclei stain To-Pro-3 Iodide Nucleic Acid Stain (Cat#T3605, Thermo Fisher). Cells were then rinsed twice with PBS and imaged using a Carl Zeiss Laser Scanning Microscope 700 with lasers at 488 nm and 639 nm and corresponding filter sets.
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