Cells harvested from QES were fixed and permeabilized using the BD Cytofix/Cytoperm™ kit, according to the manufacturer's instructions. Briefly, cells were resuspended and incubated in 250 μl of BD Cytofix/Cytoperm solution for 20 min at 4°C, and after centrifugation (300 × g for 5 min), the cells were resuspended in BD Perm/Wash buffer for 30 min at 4°C. Cells were stained with Alexa Fluor® 488 mouse anti-Oct3/4, PE mouse anti-human Nanog, Alexa Fluor® 647 mouse anti-Sox2, PE mouse anti-SSEA-4, FITC mouse anti-human TRA-1-60, and Alexa Fluor® 647 mouse anti-human TRA-1-81 (BD). Isotypes were used as negative controls. Samples were analyzed using BD LSRFortessa and FlowJo v10 software.
Alexa fluor 488 mouse anti oct3 4
Manufactured by BD
Sourced in United Kingdom, United States
Alexa Fluor® 488 mouse anti-OCT3/4 is a fluorescently-labeled monoclonal antibody that specifically binds to the OCT3/4 (octamer-binding transcription factor 3/4) protein. OCT3/4 is a critical regulator of embryonic stem cell pluripotency and self-renewal. This antibody can be used to detect and quantify OCT3/4 expression in various cell and tissue samples through techniques such as flow cytometry, immunofluorescence, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Pe mouse anti ssea 4, by BD (2 mentions)
Fitc mouse anti human tra 1 60, by BD (2 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Alexa fluor 647 mouse anti sox2, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Hoechst 33342, by Lonza (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti afp, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde solution, by Fujifilm (1 mentions)
Permeabilization wash buffer, by BD (1 mentions)
Alexa fluor 647 mouse anti ssea 4, by BD (1 mentions)
Vi cell xr, by Beckman Coulter (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Cytofix cytoperm plus fixation permeabilization kit, by BD (1 mentions)
4 protocols using alexa fluor 488 mouse anti oct3 4
1
Pluripotent Stem Cell Characterization
2
Multimarker Immunofluorescence Staining Protocol
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Samples were washed with PBS, fixed with 4% paraformaldehyde solution (Wako Pure Chemical Industries) for 20 min, permeabilized with 1% Triton™ X-100 (Sigma-Aldrich) in PBS for 20 min, and then stained overnight with the following antibodies: phycoerythrin (PE) mouse anti-human NANOG, Alexa Fluor® 488 mouse anti-OCT3/4 (BD Biosciences), mouse anti-TUJ1 (β-tubulin III; Sigma-Aldrich), rabbit anti-α-SMA (PA1-37024, Thermo Fisher Scientific), and rabbit anti-AFP (Abcam, Cambridge, UK). The secondary antibodies Alexa Fluor® 488 goat anti-mouse IgG (Thermo Fisher Scientific) and Alexa Fluor® 568 Goat Anti-Rabbit IgG (Thermo Fisher Scientific) were then added. The nuclei were stained with Hoechst 33342 (Lonza).
3
Characterization of Human iPSCs by Flow Cytometry
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Cells were dissociated with the Accutase cell detachment solution for 5 min. The dissociated human iPS cells were washed with PBS, fixed with 4% paraformaldehyde solution for 20 min, permeabilized with permeabilization/wash buffer (BD Biosciences) for 20 min, and then stained with PE mouse anti-SSEA-4, FITC mouse anti-human TRA-1-60, and Alexa Fluor® 488 mouse anti-OCT3/4 (BD Biosciences) for 1 h on ice. Stained cells were then analysed using the FACSVerse™ and FACSuite™ software.
4
Characterization of hiPSC Cultures
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During passaging, numbers of viable cells in culture vessels were determined using the trypan blue exclusion test measured by automated counting of detached cells (Vi-CELL™ XR, Beckman Coulter, Brea, CA, USA). Cumulative population doubling at each passage was calculated from the number of viable cells in each passage.
To analyze surface marker expression by flow cytometry, hiPSCs were harvested and processed according to the manufacturer's instructions. In brief, cells were detached from culture surfaces and fixed with Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences, San Diego, CA, USA) at 4 °C for 20 min, washed twice with BD Perm/Wash Buffer, then stained with Alexa Fluor® 647 mouse anti-SSEA-4 and Alexa Fluor 488 mouse anti-OCT3/4 (BD Biosciences) for 20 min in the dark. After two washes with BD Perm/Wash Buffer, stained cells were analyzed on a BD Accuri C6 flow cytometer.
For evaluation of karyotype stability, hiPSCs grown for 32 passages were assessed using a standard G-banding technique. Chromosomes were prepared using standard protocols, G-banded with trypsin and stained with Giemsa. For each culture, 20 metaphase spreads were examined.
To analyze surface marker expression by flow cytometry, hiPSCs were harvested and processed according to the manufacturer's instructions. In brief, cells were detached from culture surfaces and fixed with Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences, San Diego, CA, USA) at 4 °C for 20 min, washed twice with BD Perm/Wash Buffer, then stained with Alexa Fluor® 647 mouse anti-SSEA-4 and Alexa Fluor 488 mouse anti-OCT3/4 (BD Biosciences) for 20 min in the dark. After two washes with BD Perm/Wash Buffer, stained cells were analyzed on a BD Accuri C6 flow cytometer.
For evaluation of karyotype stability, hiPSCs grown for 32 passages were assessed using a standard G-banding technique. Chromosomes were prepared using standard protocols, G-banded with trypsin and stained with Giemsa. For each culture, 20 metaphase spreads were examined.
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