Cells were stained for 1 h at 4 °C with primary antibodies and immunofluorescent secondary antibodies. The cells were then analyzed on a Cytomics FC 500 (Beckman Coulter, Inc.) and the data were analyzed with the FlowJo Ver. 7 (Tree Star, Inc.). Antibodies against human Globo H (ALX-804-550C050, Enzo Life Sciences, Inc.) and H antigen (anti-blood group H1 (O) antigen antibody:ab3355, abcam) were adopted as primary antibodies. Alexa Fluor 488 goat anti-mouse IgM (μ chain, A21042, Invitrogen) and Alexa Fluor 488 F(ab’)2 fragment goat anti-mouse IgG (H + L) (A11017, Invitrogen) were used as secondary antibodies when the primary antibodies were to Globo H and H antigen, respectively. Fluorescent-conjugated antibodies (555401, BD Pharmingen and FAB1435F, R&D) were used for the analyses of SSEA-1 and SSEA-4, respectively. Isotype antibodies were used as control (Supplemental Table S5C ).
Fab1435f
Manufactured by R&D Systems
FAB1435F is a compact laboratory centrifuge designed for general-purpose applications. It features a fixed-angle rotor that can accommodate various sample tubes and microplates. The centrifuge operates at a maximum speed of 6,000 RPM, providing a relative centrifugal force (RCF) of up to 3,000 x g.
Automatically generated - may contain errors
Lab products found in correlation
Microcentrifuge tube, by Avantor (3 mentions)
Laser scanning microscope 700, by Zeiss (3 mentions)
Fc001, by R&D Systems (3 mentions)
Fab4770p, by R&D Systems (3 mentions)
To pro 3 iodide nucleic acid stain, by Thermo Fisher Scientific (3 mentions)
Fc005, by R&D Systems (3 mentions)
Mabd24a4, by Merck Group (3 mentions)
Ab3355, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse igm μ chain, by Thermo Fisher Scientific (1 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
4 protocols using fab1435f
1
Flow Cytometric Analysis of Stem Cell Markers
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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