Cells plated on a glass-bottom dish (AGC Techno Glass Inc., Shizuoka, Japan) were treated with 25 nM TMRM, which reflects mitochondrial membrane potential, for 30 min. The cells were then washed three times with PBS and visualized by high-resolution confocal microscopy (SP8; Leica). The intensity of TMRM was quantified using ImageJ software.
Glass-bottom dish
Manufactured by AGC Techno Glass
Sourced in Japan
The Glass-bottom dish is a laboratory equipment designed for various applications in cell culture and microscopy. It features a transparent glass bottom that allows for direct observation and imaging of cells or specimens under a microscope. The dish provides a controlled environment for sample cultivation and examination.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488 or 546, Thermo Fisher Scientific (3 mentions)
Triton X-100, Merck Group (3 mentions)
αSMA A2547, Merck Group (2 mentions)
Ab187881, Abcam (2 mentions)
LSM 510 confocal laser scanning microscope, Zeiss (2 mentions)
Ab216875, Abcam (2 mentions)
OCT4 (C-10,, Santa Cruz Biotechnology (2 mentions)
MAB1435, R&D Systems (2 mentions)
TRA-1-60, Merck Group (2 mentions)
Nanog, ReproCELL (2 mentions)
MAB1924, R&D Systems (2 mentions)
MAB4304, Merck Group (1 mentions)
BZ-X700 microscope, Keyence (1 mentions)
Gibco Dulbecco’s phosphate buffered saline (DPBS), Thermo Fisher Scientific (1 mentions)
Hoechst 33342, Nacalai Tesque (1 mentions)
FluoroBrite DMEM, Thermo Fisher Scientific (1 mentions)
BZ-X700 fluorescence microscope, Keyence (1 mentions)
Hoechst 33342, Keyence (1 mentions)
MAB2155, R&D Systems (1 mentions)
5 protocols using Glass-bottom dish
1
Mitochondrial Membrane Potential Imaging
2
Immunofluorescence Staining of Stem Cells
Cells were cultured in a glass-bottom dish (AGC Techno Glass, Shizuoka, Japan) and fixed with 4% paraformaldehyde for 20 min at 4 °C before being permeabilization with 0.1% Triton X-100 (Sigma) for 10 min at 25 °C. After blocking with 5% normal goat serum in DPBS for 30 min at RT, samples were incubated with primary antibodies at 4 °C overnight. After washing with DPBS, the samples were incubated with secondary antibodies conjugated to Alexa 488 or 546 (Thermo Fisher Scientific) for 30 min at 25 °C. After washing with DPBS, mounting medium containing DAPI was used. The following primary antibodies were used: anti-βIII tubulin (TUJ1; G712A, Promega, Madison, Wisconsin, USA), α-smooth muscle cell actin (α-SMA; A2547, Sigma), SOX17 (MAB1924; R&D Systems), OCT3/4 (SC5729, Santa Cruz Biotechnology, Santa Cruz, California, USA), NANOG (RCAB004P–F, ReproCELL, Kanagawa, Japan), SSEA4 (MAB4304, Merck Millipore, Billerica, Massachusetts, USA), and TRA-1-60 (MAB4360, Merck Millipore). Images were acquired with a BZ-X700 microscope (Keyence). All antibodies, except for the anti-TUJ1 antibody (1:300), were used at a 1:150 dilution in 5% normal goat serum.
3
Immunofluorescence Staining of Stem Cells
Cells were cultured in a glass-bottom dish (AGC TECHNO GLASS, Shizuoka, Japan) and fixed with 4% paraformaldehyde for 10 min at 4 °C before being permeabilized with 0.1% Triton X-100 (Sigma) for 10 min at room temperature (RT). After blocking with 5% normal goat serum in Gibco™ Dulbecco's phosphate-buffered saline (DPBS; Thermo Fisher Scientific) for 30 min at RT, samples were incubated with primary antibodies at 4 °C overnight. After washing with DPBS, samples were incubated with secondary antibodies conjugated to Alexa 488 or 546 (Thermo Fisher Scientific) for 30 min at RT. After washing with DPBS, mounting medium with DAPI was used.
Primary antibodies specific for the following proteins were used in this study: OCT4 (C-10; Santa Cruz Biotechnology, Dallas, TX), NANOG (ReproCell), Tra 1-60 (MAB4360; Sigma–Aldrich), SSEA4 (MAB1435, R&D Systems), KLF4 (ab216875; Abcam), PRDM14 (ab187881; Abcam), anti-βIII Tubulin (TUJ1, Promega, Madison, Wisconsin), α-smooth muscle cell actin (α-SMA; A2547; Sigma), SOX17 (MAB1924; R&D Systems). Images were acquired using an LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). All antibodies, except for the anti-TUJ1 antibody (1:300), were used at a 1:150 dilution in 5% normal goat serum.
Primary antibodies specific for the following proteins were used in this study: OCT4 (C-10; Santa Cruz Biotechnology, Dallas, TX), NANOG (ReproCell), Tra 1-60 (MAB4360; Sigma–Aldrich), SSEA4 (MAB1435, R&D Systems), KLF4 (ab216875; Abcam), PRDM14 (ab187881; Abcam), anti-βIII Tubulin (TUJ1, Promega, Madison, Wisconsin), α-smooth muscle cell actin (α-SMA; A2547; Sigma), SOX17 (MAB1924; R&D Systems). Images were acquired using an LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). All antibodies, except for the anti-TUJ1 antibody (1:300), were used at a 1:150 dilution in 5% normal goat serum.
4
Mitochondrial Membrane Potential Analysis
Primary cardiomyocytes and hepatocytes were plated on a glass bottom dish (AGC Techno Glass, Tokyo, Japan) and type I collagen-coated glass-bottom chamber (Matsunami Glass Ind., Tokyo, Japan), respectively. Cells were treated with 2 μM JC-1 (Thermo Fisher Scientific, CA, USA) and 2 μM Hoechst 33342 (Nacalai Tesque Inc., Kyoto, Japan) in HBSS for 30 min, washed twice with FluoroBrite DMEM (Thermo Fisher Scientific, CA, USA), then incubated with FluoroBrite DMEM with or without Na2S4 for 45 min. The JC-1 and Hoechst 33342 signals were measured using a BZ-X700 fluorescence microscope (Keyence, Osaka, Japan) and the red/green ratio of the acquired images was quantified.
5
Immunofluorescence Staining of Stem Cells
Cells were cultured in a glass-bottom dish (AGC Techno Glass, Shizuoka, Japan) and fixed with 4% paraformaldehyde for 10 min at 4 °C before being permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature (RT). The samples were blocked with 5% normal goat serum in Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific) for 30 min at RT. They were incubated with primary antibodies at 4 °C overnight. After washing with DPBS, the samples were incubated with secondary antibodies conjugated to Alexa 488 or 546 (Thermo Fisher Scientific) for 30 min at RT. After washing with DPBS, the samples were placed in mounting medium with 4',6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: OCT4 (C-10; Santa Cruz Biotechnology, Dallas, TX, USA), NANOG (ReproCell), Tra1-60 (MAB4360; Sigma-Aldrich), SSEA4 (MAB1435; R&D Systems), SSEA1 (MAB2155; R&D Systems), KLF4 (ab216875; Abcam), PRDM14 (ab187881; Abcam), anti-βIII Tubulin (TUJ1, Promega, Madison, Wisconsin, USA), α-smooth muscle actin (α-SMA; A2547; Sigma-Aldrich), and SOX17 (MAB1924; R&D Systems). Images were acquired using an LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). All antibodies except for the anti-TUJ1 antibody (1:300) were used at a 1:150 dilution in 5% normal goat serum.
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