According to the manufacturer’s instructions, MitoTracker Red (Cat# M22425, Invitrogen, Carlsbad, CA, USA) and ER-Tracker Blue (Cat# E12353, Invitrogen, Carlsbad, CA, USA) were used to observe the morphological changes of mitochondrial and ER, respectively. In brief, SCs were stained with MitoTracker Red or ER-Tracker Blue for 30 min at 37 °C, and the images were captured under a fluorescence microscope (Olympus BX53, Tokyo, Japan).
Er tracker blue
Manufactured by Thermo Fisher Scientific
Sourced in United States
ER-Tracker Blue is a fluorescent dye that specifically labels the endoplasmic reticulum (ER) in live cells. It can be used to visualize the ER structure and dynamics in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Mitotracker red, by Olympus (1 mentions)
Glass bottom fluorodishes, by World Precision Instruments (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
A31571, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Fitc conjugated goat anti mouse immunoglobulin g igg, by Proteintech (1 mentions)
Cy3 conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Rabbit anti atg5 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti p62 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti lc3 antibody, by Merck Group (1 mentions)
Mouse anti hcv ns5a monoclonal antibody, by Merck Group (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Rabbit anti calnexin antibody, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Polyvinylidene difluoride pvdf membrane, by GE Healthcare (1 mentions)
Ab21685, by Abcam (1 mentions)
4 pba, by Merck Group (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
6 protocols using er tracker blue
1
Visualizing Mitochondrial and ER Dynamics
2
Visualizing Intracellular Organelles in HeLa Cells
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Cells were plated, at a density of 3000 cells/cm2, on glass-bottom fluorodishes (World Precision Instruments) coated with collagen. Cells were transfected 48 h before imaging using Leica SP5 confocal microscope with a 63X objective. Nuclei were labeled with Hoechst staining (blue). A TMEM33-EGFP construct was stably expressed in Hela cells. Primary cilia were visualized with acetylated tubulin antibody (Sigma T7451; 1/200) and a secondary donkey anti-mouse antibody (Alexa 647 Invitrogen A-31571; 1/500). ER was marked with ER Tracker blue (invitrogen E12353) and lysosomes by Lysotracker red (invitrogen L7528).
3
EV71 Infection and Immunofluorescence Microscopy
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RD cells were seeded on 20-mm cover slips to 90% confluence and infected with EV71 at an MOI of 5. At 6 or 12 h p.i., culture medium was removed and cells were washed three times with PBS and fixed with 3.7% formaldehyde for 20 min at room temperature. Cells were then washed with PBS and permeabilized using 0.4% Triton X-100 for 5 min at room temperature. After another PBS wash, cells were blocked in PBS containing 5% BSA for 1 h at 37°C, incubated with antibodies for 3 h at 37°C and washed three times with PBS. The samples were incubated with FITC-conjugated goat anti-mouse immunoglobulin G (IgG) and Cy3-conjugated goat anti-rabbit IgG (ProteinTech Group) for 45 min at room temperature. For live-cell ER labeling, 1 µM ER-Tracker™ Blue (Invitrogen) stained cells for 15 min at 37°C. To stain nuclei, 1 µg/ml DAPI (Roche) methanol solution was added and samples incubated for 15 min at room temperature. After washing with PBS, samples were visualized by confocal laser-scanning microscopy (Fluoview FV1000; Olympus, Tokyo, Japan).
4
Immunoblotting of Autophagy Proteins
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The primary antibodies used in this study included the mouse anti-STX7 antibody (Sigma-Aldrich), rabbit anti-ATG5 antibody (Cell Signaling), rabbit anti-p62 antibody (Cell Signaling), mouse anti-HCV NS5A monoclonal antibody (Millipore), rabbit anti-LC3 antibody (Sigma-Aldrich), and rabbit anti-calnexin antibody (Abcam). Proteins were extracted from cell lysates for western-blot analysis using the M-PER mammalian protein extraction reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. The ER Tracker Blue was purchased from Thermo Fisher Scientific.
5
Mitochondrial Dynamics Regulation in ERS
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This study used monoclonal antibodies (mAb) against Mitofusin1 (Mfn1, #14,739), Mitofusin2 (Mfn2, #11,925), Optic atrophy 1 (OPA1, #67,589), Mitochondrial fission factor (MFF, #84,580), Dynamin-related protein 1 (Drp1, #8570), p-Drp1 Ser616 (#4494, Cell Signaling Technology, Beverly, MA, USA); Glucose-regulated protein 78 (GRP78, ab21685) and β-actin (ab8226, Abcam. Cambridge, MA, USA); polyclonal antibodies (pAb) against GRP75 (A0558), C/EBP homologous protein (CHOP, A0221, Abclonal, Wuhan, China). Other specific reagents included MitoTracker® Deep Red FM (M22426) and ER-Tracker™ Blue (E12353), bicinchoninic acid (BCA) protein detection kit (#23,227, Thermo Fisher Scientific, Waltham, MA, USA), polyvinylidene difluoride (PVDF) membrane (GE Health, New York, USA), DCFH-DA (D6883) and 4-phenylbutyrate (4-PBA, an ERS inhibitor, SML0309, Sigma, St. Louis, USA); DAPI (G1012, Servicebio, Wuhan, China), JC-1 mitochondrial membrane potential detection (MMP) kit (C2006) and ATP detection kit (S2006, Beyotime Biotechnology, Beijing, China), MKT077 (a GRP75 inhibitor, HY15096, MCE, NJ, USA), and GRP75 siRNA (AuGCT Biotechnology, Wuhan, China).
6
Investigating NET-mediated T cell Cytotoxicity in HCC
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1 × 105 HepG2 cells were seeded in 12-well plate coated with 0.5 ml of ECM (10 mg/ml), ECM-Col1 (10 mg/ml), Col1 (1 mg/ml) or PBS, after formation of small clonality, peripheral neutrophils from HCC patients were co-cultured with tumor cells at 20:1 ratio for 4 h for allowance to extrude NETs. Then neutrophils were gently removed by PBS washing, leaving tumor cells covered by different amounts of NETs. Then 2 × 106 aCD3 plus aCD28 (Biolegend) activated CD8+ T cells from PBMC were added to the culture assay and co-cultured for 2 days, to test how NETs affect T cells cytotoxicity.
Tumor cell survival was examined by flow cytometer. After co-cultured with T cells, suspended immune cells were gently removed with PBS. Then cells were collected with Trypsin and labeled with CSFE (Thermo) and then fixed with 4% paraformaldehyde. Then anti-CD45 antibody (BD-562886, BD) was used to mark immune cells in assay. Then flow cytometer was used to detect tumor cell survival rate. Live tumor cells were marked as CD45−CSFEhighConfocal speculation was performed 10 h after T cell address. Tumor cells and T cells were marked with ER tracker blue (E12353, Thermo) or DiO (D3911, Thermo) before added into co-culture assay. NETs were marked with SytoxGreen (S7020, Invitrogen).
Tumor cell survival was examined by flow cytometer. After co-cultured with T cells, suspended immune cells were gently removed with PBS. Then cells were collected with Trypsin and labeled with CSFE (Thermo) and then fixed with 4% paraformaldehyde. Then anti-CD45 antibody (BD-562886, BD) was used to mark immune cells in assay. Then flow cytometer was used to detect tumor cell survival rate. Live tumor cells were marked as CD45−CSFEhighConfocal speculation was performed 10 h after T cell address. Tumor cells and T cells were marked with ER tracker blue (E12353, Thermo) or DiO (D3911, Thermo) before added into co-culture assay. NETs were marked with SytoxGreen (S7020, Invitrogen).
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