Mitochondrial health was assessed using the fluorescent dye tertramethyl rhodamine ethyl ester (TMRE): brighter fluorescence indicates healthier mitochondria. Intracellular Ca2+ levels were assessed using Oregon Green® 488 BAPTA-1 AM. First, cells were incubated with different concentrations of ziram in DMEM medium. After ziram treatment, BHK cells were loaded with 20 nM TMRE and 5 μM Oregon Green BAPTA-1 for 30 min in a buffer containing (in mM): 156 NaCl, 3 KCl, 2, MgSO4, 1.25 KH2PO4, 2 CaCl2, 10 glucose, and 10 Hepes, pH adjusted to 7.35 with NaOH. At the end of the incubation, cells were washed in the buffer and examined immediately with an Imager M2 fluorescence microscope (Carl Zeiss, Germany). Stained cells were examined immediately with a Zeiss Imager M2 fluorescence microscope (Carl Zeiss, Germany) microscope equipped with Zeiss filter sets 038 (excitation BP 470/40 nm, beam splitter FT 495 nm and emission BP 525/50 nm) and 043 (excitation BP 545/25 nm, beam splitter FT 570 nm and emission BP 605/70 nm) (Carl Zeiss, Germany) allowing the specific detection of Oregon Green BAPTA (excitation peak 490/emission peak 517 nm) and TMRE (excitation peak 552/emission peak 578 nm), respectively. Fluorescence intensity was quantified using ImageJ software (Schneider et al., 2012 (link)).
Imager m2 fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Imager M2 is a fluorescence microscope designed for high-resolution imaging of biological samples. It features a multi-channel detection system, allowing for the simultaneous observation of multiple fluorescent labels. The microscope is equipped with advanced optics and a high-sensitivity camera to capture detailed images of cellular structures and dynamics.
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Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Imager m2, by Zeiss (1 mentions)
Lysotracker, by Zeiss (1 mentions)
Axioimager software, by Zeiss (1 mentions)
Mitotracker green dye, by Thermo Fisher Scientific (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Axiocam icm1, by Zeiss (1 mentions)
Acridine orange, by Zeiss (1 mentions)
Mitotracker, by Zeiss (1 mentions)
Texas red labeled dextran, by Thermo Fisher Scientific (1 mentions)
Synergy 2 microplate reader, by Agilent Technologies (1 mentions)
24 well transwell insert, by BD (1 mentions)
B 5 1 2, by Merck Group (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
A10036, by Thermo Fisher Scientific (1 mentions)
F1804, by Thermo Fisher Scientific (1 mentions)
A10040, by Thermo Fisher Scientific (1 mentions)
R960 25, by Merck Group (1 mentions)
8 protocols using imager m2 fluorescence microscope
1
Assessing Mitochondrial Health and Calcium Levels
2
Immunohistochemical Analysis of Epithelial and Mesenchymal Markers in MDA-MB-231 Tumors
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The presence of the characteristic human epithelial marker, E-cadherin, and human mesenchymal marker, N-cadherin, in human MDA-MB-231 tumors was detected using immunohistochemistry and specific antibodies. Processed tumors were embedded in paraffin blocks following necropsy in each experiment. Microtome sections of tumor at 5 μm thickness were deparaffinized, heated for 8 minutes in citrate buffer, and rinsed three times in 1× PBS, followed by blocking with 1% bovine serum albumin (Thermo Fisher Scientific) in 1× PBS overnight at 4°C. Deparaffinized and processed tumor sections (5 μm) were immunostained with primary antibody for either 2 μg/mL rabbit anti-E- or anti-N-cadherin followed by Alexa Fluor® 488 secondary donkey anti-rabbit IgG antibody. The stained cells were covered with Entellan® rapid mounting medium, and images were observed using a Zeiss Imager M2 fluorescence microscope at 200× magnification. Additional 5 μm microtome sections of tumors were analyzed for mouse endothelial marker CD31/PECAM-1 using a 1:300 dilution of a 1 mg/mL stock of rat monoclonal antibody conjugated with DyLight-488 fluorochrome against mouse CD31/PECAM-1 (Novus Biologicals Canada ULC) and a Zeiss Imager M2 fluorescence microscope.
3
BCR-ABL1 Construct Identification by FISH
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The analysis by FISH was performed essentially as described [46 (link)]. Briefly, a digoxigenin-12-dUTP labelled pITR-ETP-BCR-ABL1 plasmid was used as hybridization probe to determine the number of BCR-ABL1 construct insertions in the genome. Sheep antidigoxigenin-fluorescein isothiocyanate 1:100 (Roche Diagnostics, Basel, Switzerland) was used for immunodetection of hybridized probes. Signals were analyzed using a ZEISS Imager M2 fluorescence microscope (Carl Zeiss, Jena, Germany) and the Genesis software (Applied Spectral Imaging, Carlsbad, CA, USA).
4
Fluorescent Probes for Cellular Compartment Analysis
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Fluorescent probes that stained different cellular compartments were used to characterize the origin of the vacuoles. The dapoxyl ER-tracker blue-white dye, the Lyso-tracker red dye, the Mito-tracker green dye, and 10 kDa and 70 kDa Texas-Red labeled dextran were all obtained from Molecular Probes (Life Technologies, Merelbeke, Belgium). We also used Lucifer yellow (Lucifer Yellow CH, lithium salt) from Biotium (Fremont, CA, USA) and acridine orange from Sigma-Aldrich. Briefly, the cell seeding and treatment procedures were similar to the ones used for the phase contrast microscopy (Section 4.3.1 .). The dye solutions were simply added to the culture medium 1 h before the end of the treatment periods, excepted for the Lucifer yellow and both dextrans 10 kDa and 70 kDa, which were added for the whole duration of the treatment. The concentrations of the dyes were as follows: ER tracker, 0.5 µM; Lyso tracker, 75 nM; Mito tracker, 200 µM; Lucifer yellow, 100 µg/mL; acridine orange, 1 µg/mL; and dextrans 10 kDa and 70 kDa, 125 µg/mL; At the end of the treatment period, the procedure was similar to that of phase-contrast microscopy to take pictures of living cells with the Imager M2 fluorescence microscope (Carl Zeiss) coupled with the AxioCam ICm1 and AxioImager software (Carl Zeiss). The experiment was realized at least two times in duplicate.
5
Cell Proliferation and DNA Replication Assays
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Cellular proliferation was assessed using Cell Counting Kit‐8 (CCK‐8) assays as we previously described.22 Briefly, 2000 indicated cells resuspended in 100 μL complete medium were seeded into a 96‐well plate. After culture for the indicated time, 10 μL CCK‐8 reagent (cat. no. CK04, Dojindo) was added to each well. After culture for another 2 h, the absorbance values at 450 nm were detected using the Synergy 2 microplate reader (BioTek) to indicate the viable cell number. DNA replication was assessed using 5‐ethynyl‐2′‐deoxyuridine (EdU) incorporation assays. EdU incorporation assays were performed using the Cell‐Light EdU Apollo567 In Vitro Kit (cat. no. C10310‐1, RiboBio). The percentage of EdU‐positive cells was detected using the Imager.M2 fluorescence microscope (Carl Zeiss) and calculated as the ratio of EdU‐positive cells to total cells.
6
Transwell Invasion Assay Protocol
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Indicated cells suspended in serum free medium were plated into the upper chamber of 24-well transwell inserts (8-μm pore size, BD Biosciences, San Jose, CA, USA) at 50,000 cells/well. Medium containing 20% bovine fetal serum was added to the lower chamber. After culture for 48 h, the cells remaining in the upper chamber were removed and the cells on the lower surface were fixed, stained, and counted using the Imager.M2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
7
Comprehensive Gene Expression Analysis
8
Immunofluorescent Localization of FAD-GFP in Tetrahymena
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Tetrahymena expressing 12 FAD-GFP were fixed with 4% (w/v) paraformaldehyde in 50 mM HEPES pH 7.0 for 10 min at room temperature, washed with ice-cold HEPES two times and permeabilized with ice-cold 0.2% Triton X-100 in 50 mM HEPES pH 7.0 for 8 min. After being washed with ice-cold HEPES three times, the fixed cells were treated with blocking solution (1%
[w/v] bovine serum albumin [BSA] in TBS buffer) for 1 h at room temperature, and incubated for 1 h in blocking solution with anti-GFP primary antibody (A-11122; Invitrogen) at a 1:200 dilution.
Cells were washed three times for 5 min each with TBS buffer containing 0.1% (w/v) BSA and subsequently incubated for 1 h at room temperature with goat anti-mouse-Alexa Fluor 594 secondary antibody (A-11020; Invitrogen) at a 1:400 dilution in blocking solution. After one wash in TBS buffer containing 0.1% (w/v) BSA the cells were incubated with 1 mM DAPI (4´,6-diamidino-2phenylindole in EtOH) for 15 min at room temperature. Finally, cells were washed twice with 50 mM HEPES pH 7.0 and mounted with Trolox antibleaching solution. Digital images were collected using a Carl Zeiss Axio imager M2 fluorescence microscope. Images were analyzed with Image J 1.50i (Wayne Rasband, National Institute of Health, USA).
[w/v] bovine serum albumin [BSA] in TBS buffer) for 1 h at room temperature, and incubated for 1 h in blocking solution with anti-GFP primary antibody (A-11122; Invitrogen) at a 1:200 dilution.
Cells were washed three times for 5 min each with TBS buffer containing 0.1% (w/v) BSA and subsequently incubated for 1 h at room temperature with goat anti-mouse-Alexa Fluor 594 secondary antibody (A-11020; Invitrogen) at a 1:400 dilution in blocking solution. After one wash in TBS buffer containing 0.1% (w/v) BSA the cells were incubated with 1 mM DAPI (4´,6-diamidino-2phenylindole in EtOH) for 15 min at room temperature. Finally, cells were washed twice with 50 mM HEPES pH 7.0 and mounted with Trolox antibleaching solution. Digital images were collected using a Carl Zeiss Axio imager M2 fluorescence microscope. Images were analyzed with Image J 1.50i (Wayne Rasband, National Institute of Health, USA).
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