The opening mPTP of cardiomyocytes were detected by using the calcein-cobalt with a mPTP assay kit (Genmed Scientifics, cat #GMS10095) according to the manufacture's directions. Briefly, cardiomyocytes, seeded in 24-well plates, were washed with Reagent A, then incubated with Reagent B and C (1:50; 500 μl per well) at 37 C for 20 min, then washed twice with Reagent A again. Fluorescence intensity was measured using a InfiniteTM M200 Microplate Reader (Tecan). Cells were subsequently lysed in 20 μl of 0.1 M NaOH and protein concentration was measured using the Bradford Protein assay. The fluorescent signals were normalized to total protein content in the corresponding cell extract. Results were presented as normalized relative fluorescence units (NRFU; U/mg protein).
Mptp assay kit
Manufactured by Genmed Scientifics
Sourced in China, United States
The MPTP assay kit is a laboratory tool used to detect and quantify the presence of MPTP, a compound commonly used in research. The kit provides a standardized and reliable method for measuring MPTP levels in various samples. It is designed to assist researchers in their scientific investigations, without making any claims about its intended use or applications.
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Lab products found in correlation
Infinite m200 microplate reader, by Tecan (1 mentions)
Caspase 3 antibody, by Beyotime (1 mentions)
Bcl 2 antibody, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Resveratrol, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
Jc 1 probe, by Beyotime (1 mentions)
Varioskan flash, by Thermo Fisher Scientific (1 mentions)
Mitosox, by Beyotime (1 mentions)
Mitotracker, by Beyotime (1 mentions)
Mitosox, by Yeasen (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
8 protocols using mptp assay kit
1
Detecting Mitochondrial Permeability Transition Pore
2
Mitochondrial Permeability Transition Pore Assay
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The opening level of mitochondrial permeability transition pore (MPTP) in PAFs and PASMCs was measured using the MPTP assay kit (Genmed). First, cell medium was discarded, and preheated cleaning solution was added. Next, cells were incubated with working solution at 37°C and protected from light for 30 minutes. After washing 3 times with 0.01 mol/L PBS, fluorescent images were captured and analyzed using a laser scanning confocal microscope. As a fluorescent probe, calcein AM can be captured easily by mitochondria and presents with green fluorescence. When MPTP opens, calcein is released into cytoplasm, with fluorescent quenching.
3
MPTP-induced mitochondrial dysfunction assay
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The MPTP opening of NP cells was assessed by the MPTP Assay Kit (Genmed, China) as previously described [15 (link)]. After 0, 24, and 36 h compression, the cells were collected; afterward, 500 μl preheated cleaning solution (Reagent A) and isopyknic working solution containing neutralization and staining solution (Reagent B) were added into the cell suspension. Then, the above cell suspension was mixed gently and fully and incubated for 20 min at 37°C in the dark. Lastly, the samples were resuspended in Reagent A and analyzed by flow cytometry.
4
Resveratrol Cytotoxic Effects in Vitro
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Resveratrol (purity 99%; Sigma), RPMI-1640 medium, and fetal bovine serum (FBS) were obtained from Invitrogen (Shanghai, China).
MTT Cell Proliferation and Cytotoxicity Assay Kit (catalog No. C0009), Caspase-3 antibody (catalog No. AC033), Bcl-2 antibody (catalog No. AB112), secondary antibody [HRP-labeled goat anti-rabbit IgG (H+L)] (catalog No. A0208), JC-1 probe (catalog No. C2006), ATP assay kit (catalog No. S0026), and BCA protein assay kit (catalog No. P0012) were purchased from Beyotime Institute of Biotechnology (Haimen, China) and MPTP assay kit (catalog No. GMS10095.1) from Genmed Scientifics Inc. (USA) Fluorescein diacetate (FDA) was obtained from Keeasy Economic & Trade Co., Ltd. All other reagents were of analytical grade and were obtained from commercial sources.
MTT Cell Proliferation and Cytotoxicity Assay Kit (catalog No. C0009), Caspase-3 antibody (catalog No. AC033), Bcl-2 antibody (catalog No. AB112), secondary antibody [HRP-labeled goat anti-rabbit IgG (H+L)] (catalog No. A0208), JC-1 probe (catalog No. C2006), ATP assay kit (catalog No. S0026), and BCA protein assay kit (catalog No. P0012) were purchased from Beyotime Institute of Biotechnology (Haimen, China) and MPTP assay kit (catalog No. GMS10095.1) from Genmed Scientifics Inc. (USA) Fluorescein diacetate (FDA) was obtained from Keeasy Economic & Trade Co., Ltd. All other reagents were of analytical grade and were obtained from commercial sources.
5
Mitochondrial Permeability Transition Pore Assay
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The mPTP Assay Kit (Genmed, US) was used to evaluate the mPTP activity, wherein fluorescence quenching by cobalt ions increased when the mPTP activity increased. The staining and fixation of cells were performed according to the manufacturer's protocol. Briefly, the culture medium was removed, and cells were washed by PBS for 2 times. Then, 1 mL calcein AM staining solution with 10 μL CoCl2 fluorescence quenching solution were added to one well of 6-well plate and incubated at 37°C for 30 min in darkness and then replaced by fresh culture medium preheated to 37°C and incubated at 37°C for 30 min in darkness again. After washing the cells with PBS for 2-3 times, the detection buffer was added, and then cells were observed by a fluorescence microscope (Olympus IX71, JP). Five replicates were performed for each group.
6
Quantifying Mitochondrial Permeability Transition
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Opened mPTPs of the quadriceps muscle were measured by fluorexon with the mPTP assay kit (Genmed Scientifics Inc., Wilmington, DE, USA), in accordance with the manufacturer's instructions. Briefly, 0.1 mL of depurated mitochondrial suspension, containing 200 μg protein, was added into Reagent A; this mixture was incubated at 37°C for 15 min and centrifuged at 16000 × g for 5 min. The supernatant was discarded and the precipitate was resuspended with Reagent C; the fluorescence intensity of the reaction medium was determined using Varioskan Flash (Thermo Fisher Scientific), with excitation wavelength (λex) 488 nm and emission wavelength (λem) 505 nm.
7
Intracellular ROS and Mitochondrial Function
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The intracellular total and mitochondrial ROS level were measured respectively by 2’,7’-dichlorofluorescin diacetate (DCFH-DA, S0033; Beyotime, Beijing, China) and MitoSOX (40778ES50; Yeasen biotech, Shanghai, China) staining. Both of them can be rapidly oxidized to highly fluorescent compounds. The samples were stained according to the manufacturers’ instructions and detected by FACSCalibur flow cytometer (BD Biosciences). Further, the mitochondrial ROS was evaluated by the colocalization of MitoSOX and Mitotracker (C1048, Beyotime) intensity detected using Laser Scanning Microscope (ZEISS LSM780, Germany).
MMP was detected using JC-1 (C2006; Beyotime) assay kits as described previously [30] (link) and analyzed by FACSCalibur flow cytometer (BD Biosciences). The decrease ratio of red (JC-1 aggregates) to green (JC-1 monomers) indicated the MMP loss. The mPTP Assay Kit (GMS10095.1; Genmed, Shanghai, China) was used to detect mPTP activity, wherein fluorescence quenching by cobalt ions increased when the mPTP activity increased. NP cell staining and fixation were performed according to the manufacturer's instructions. Next, the slides were observed and imaged using a fluorescence microscope (Olympus IX71).
MMP was detected using JC-1 (C2006; Beyotime) assay kits as described previously [30] (link) and analyzed by FACSCalibur flow cytometer (BD Biosciences). The decrease ratio of red (JC-1 aggregates) to green (JC-1 monomers) indicated the MMP loss. The mPTP Assay Kit (GMS10095.1; Genmed, Shanghai, China) was used to detect mPTP activity, wherein fluorescence quenching by cobalt ions increased when the mPTP activity increased. NP cell staining and fixation were performed according to the manufacturer's instructions. Next, the slides were observed and imaged using a fluorescence microscope (Olympus IX71).
8
Assessing Mitochondrial Permeability Transition
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The mPTP opening was detected by using an mPTP assay kit (Genmed Scientifics) based on the calcein‐AM/cobalt method following the manufacturer's instructions. Neurons were plated in 24‐well plates (5 × 104 cells per well). After the designated treatments, neurons were rinsed twice with PBS and the staining was done by incubating with calcein‐AM (1 μM per well) and cobalt dichloride (1 mM per well) for 20 min at 37 °C. The intensities of fluorescence were assessed at 488 nm for excitation and at 505 nm for emission. The mPTP opening of each experimental group was represented by normalized fluorescence to protein concentration.
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