In order to detect mitochondrial distribution, the denuded living oocytes were moved into Mito-Tracker green (200 nM, Beyotime, Shanghai, China) diluted in M2 medium for 30 min at 37°C in dark. The mitochondrial membrane potential was measured with Mitochondrial Membrane Potential Assay Kit with TMRE (C2001S, Beyotime); the tetramethylrhodamine, ethyl ester (TRME) can gather in matrix of polar mitochondria with orange fluorescence, and there is no TMRE accumulation in lower membrane potential of mitochondria. The cumulus-denuded oocytes were stained with TMRE (1:1000) for 20 min away from light. For ER staining, it is 30 min for the living oocytes incubating in ER tracker green (C1042S, 1:1000, Beyotime). For staining DNA of living oocytes, the oocytes were cultured with Hoechst 33342 (C1025, Beyotime) for 10 min. Finally, the samples were scanned with a Zeiss Axio Observer D1 microscope (Carl Zeiss, Inc., Thornwood, NY). It was keeping the same for the procedure of staining and settings of microscope in each group.
Er tracker green
Manufactured by Beyotime
Sourced in China
ER-Tracker Green is a fluorescent dye that selectively labels the endoplasmic reticulum (ER) in live cells. It is a tool for visualizing and studying the ER structure and dynamics within the cellular environment.
Automatically generated - may contain errors
Lab products found in correlation
Mito tracker green, by Beyotime (2 mentions)
Golgi tracker green, by Beyotime (2 mentions)
Lysotracker green, by Beyotime (2 mentions)
Mitotracker red, by Beyotime (2 mentions)
Axio observer d1 microscope, by Zeiss (1 mentions)
Mitochondrial membrane potential assay kit with tmre, by Beyotime (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Jc 1 kit, by Yeasen (1 mentions)
Fluorescent labeled monoclonal antibodies, by BioLegend (1 mentions)
αpd 1 antibody, by BioXCell (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Keygen Biotech (1 mentions)
Cell counting kit 8 cck 8, by Yeasen (1 mentions)
Mito tracker red cmxros, by Beyotime (1 mentions)
Confocal microscope, by Nikon (1 mentions)
Amiloride, by Merck Group (1 mentions)
Chlorpromazine, by Merck Group (1 mentions)
Lovastatin, by Merck Group (1 mentions)
Mouse anti rab7, by Abcam (1 mentions)
2 4 6 trinitrobenzenesulfonic acid tnbs, by Merck Group (1 mentions)
8 protocols using er tracker green
1
Mitochondrial and ER Dynamics in Oocytes
2
Comprehensive ROS-sensing Protocol
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Phosphate-Buffered Saline (PBS, pH 7.4), penicillin–streptomycin, and ROS detecting probe were purchased from Keygen (Nanjing, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, and trypsin were purchased from Thermo Fisher Scientific (MA, USA). Cell Counting Kit 8 (CCK-8), Annexin V-PI cell apoptosis kit, and JC-1 kit were purchased from Yeasen Biotechnology (Shanghai, China). Thiophene-2-thiol,4,6-dia-midino-2-10 phenylindole (DAPI), Mito-Tracker green, Golgi-Tracker green, ER-Tracker green, and Lyso-Tracker green were purchased from Beyotime (Shanghai, China). All fluorescent-labeled monoclonal antibodies were obtained from Biolegend (CA, USA) and Beyotime (Shanghai, China). αPD-1 antibody was supplied from BioXcell (NH, USA) and Zinc Phthalocyanine (ZnPc, 98%) was purchased from Adamas (Shanghai, China). All other chemical reagents were purchased commercially from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
3
Visualizing ER-Mitochondria Interactions
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H9c2 cells were incubated with Mito-Tracker Red CMXRos (C1035; Beyotime Biotechnology, China) and ER-Tracker Green (C1042S; Beyotime Biotechnology, China) for 30 min at 37°C, and washed with prewarmed Hanks' balanced salt solution (HBSS, C0219; Beyotime Biotechnology, China), then fixed with 4% paraformaldehyde for 2 min at 37°C. The contact of ER and mitochondria was observed with a confocal microscope (Nikon, Tokyo, Japan) using a 100x oil immersion lens. ER and mitochondria contact analysis was measured on Image J using the Pearson's correlation coefficient.
4
Colocalization of ER and Mitochondria
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The cells were labeled with MitoTracker Red (Beyotime) and ER-Tracker Green (Beyotime) and observed using a confocal microscope (Olympus AX70, Tokyo, Japan). Pearson correlation analysis was used to quantify the degree of colocalization between the fluorophore ER and mitochondria.32 (link) Pearson's correlation coefficient was analyzed using the colocalization analysis module from ImageJ software (NIH).
5
Fluorescent Nanoparticle-Based Cellular Assays
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Fluorescent (Ex 620, Em 680 nm) polystyrene nanoparticles (PS) with a theoretical diameter of 100 nm were purchased from Tianjin BaseLine ChromTech Research Centre (Tianjin, China). Streptozotocin (STZ) and Hank's balanced salt solution (HBSS) were purchased from Solarbio Life Sciences (Beijing, China). TNBS (2, 4, 6-trinitrobenzene sulfonic acid), amiloride, chlorpromazine, and lovastatin were purchased from Sigma–Aldrich (St. Louis, MO, USA). Glycine sarcosine (Gly-Sar) was purchased from Tokyo Chemical Industry Co., Led. (Tokyo, Japan). Ezetimibe was purchased from Huaxia Chemical Reagents Co., Ltd (Chengdu, China). BCA protein assay kit, LDH assay kit, Reactive oxygen species (ROS) assay kit, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), 4ʹ,6-diamidino-2-phenylindole (DAPI), Lyso-Tracker Green, ER-Tracker Green, Golgi-Tracker Green were purchased from Beyotime Biotechnology (Shanghai, China). Fluo-4 AM was purchased from Meilun Biotechnology (Dalian, China). Rabbit anti-rab5, mouse anti-rab7, rabbit anti-rab11, rabbit anti-claudin-1, and Alexa Fluor 488 labeled goat anti-rabbit/mouse IgG were purchased from Abcam (Cambridge, UK). Anti-VAMP8 antibody and anti-Annexin A2 antibody were purchased from HUABIO (Hangzhou, China). All other chemical reagents in this study were analytic grade.
6
Intracellular Localization of ER
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HAP1 and HepG2 cells were plated into the culture dish until ~50% confluence and incubated with ICG (#H20055881, Dandong Pharmaceutical Company) for 12 h. Cells were washed with PBS three times and ER was labeled with ER-tracker green according to the manufacturer’s protocol (#C1042S, Beyotime), and the nucleus was stained blue by 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were obtained by confocal laser scanning microscopy.
7
Visualizing ER-Mitochondria Interactions
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To detect ER-mitochondria interactions, cells were incubated with ER-Tracker Green (1 μM, Beyotime, China) for 20 min, followed by MitoTracker Red (200 nM, Beyotime, China) for 30 min. After being washed thrice, the fluorescence was observed under a laser scanning confocal microscope (40×, Leica, Germany).
8
Organelle Marker Antibodies and Stains
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Mouse anti-Tom20 antibody (Cat# sc-17764) and rabbit anti-GAPDH antibody (Cat# sc25578) were obtained from Santa Cruz Biotechnology (Dallas, TX, United States). Rabbit anti-GRP78 antibody (Cat# ab21685) was purchased from Abcam (Cambridge, United Kingdom). Rabbit anti-RAB11A antibody (Cat# 2413), rabbit anti-LC3B (D11) XP (Cat# 3868), and rabbit anti-LC3 (Cat# 2775) antibody were purchased from Cell Signaling Technology (Danvers, MA, United States). Goat anti-rabbit peroxidase-conjugated secondary antibody (Cat# GTX213110-01) was purchased from Gene Tex (Irvine, CA, United States). Alexa Fluor 488 goat anti-rabbit (Cat# A32731), Alexa Fluor 488 donkey anti-mouse (Cat# A-21202), Alexa Fluor 546 donkey anti-rabbit antibodies (Cat# A10040), MitoTracker Red CMXRos (Cat# M7512), and MitoProbe JC-1 assay kit (Cat# M34152) were purchased from Invitrogen (Carlsbad, CA, United States). ER-Tracker Green, Golgi-Tracker Red, and Lyso-Tracker Red were purchased from Beyotime Biotechnology (Shanghai, China). Unless noted otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States).
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