A quantitative analysis of apoptotic cells was carried out using fluorescence microscopy (NucView® caspase-3 Kit, Biotium, Fremont, CA, USA). Twenty-four hours after irradiation, the cells were washed 3 times with PBS and stained by specific dye at a concentration of 5 µM. Caspase-3-positive cells were detected by a Bio-Rad ZOE imager (Bio-Rad, Hercules, CA, USA).
Zoe imager
Manufactured by Bio-Rad
Sourced in United States
The ZOE imager is a compact, fluorescence microscope designed for cell culture imaging. It captures high-quality images of cells and allows users to monitor cellular activity and changes over time.
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Lab products found in correlation
6 protocols using zoe imager
1
Apoptosis Quantification by Microscopy
2
Immunofluorescence Staining of Capsular Fibroblasts
3
Measuring Calcium Flux in ATP2C1-Deficient hBMVECs
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For OGB1 staining in ATP2C1 knockdown cells, hBMVECs were electroporated with mock, siATP2C1, or Q747A SPCA1-FLAG as previously described. For OGB1 in untransfected hBMVECs, cells were grown in RPMI 1640 minus Ca2+ media plus 10% FBS for 2 days prior to 1.5 h pretreatment in RPMI 1640 minus Ca2+, minus serum. Where indicated, cells were treated with 10 μM BAPTA-AM or 2 mM CaCl2 during the pretreatment. hBMVECs were incubated with 1 μM OGB1 (or no dye control) for 1 h, washed twice in PBS, then imaged at 20X magnification on a Bio-Rad ZOE imager. Images were quantified with ImageJ using n ≥ 5 ROIs per image, and the intensities were background corrected using the no OGB1 stain control and normalized to the untreated control or mock samples.
4
Rotenone-induced Oxidative Stress Assay
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IMR-32 cells were seeded at 1
× 104 cells/well in 12-well plates, differentiated,
and treated with rotenone (100 nM), Yashtimadhu choorna extract (200
μg/mL), 100 nM rotenone + Yashtimadhu choorna extract cotreatment
for 48 h. The untreated cells were taken as a control. Post-treatment,
the medium was aspirated, and the cells were washed with 1× phosphate-buffered
saline (PBS) and treated with 20 μg/mL of PI for dead cells
and 5 μg/mL of HOECHST as a counter nuclear stain in serum-free
media for 15 min.75 (link) For staining of the
intracellular ROS productions, the cells were stained with DCFDA post-treatment
with rotenone, Yashtimadhu alone, and Yashtimadhu choorna extract
cotreatment. The cells were washed with 1× PBS and stained with
25 μM of DCFDA and 5 μg/mL of HOECHST in serum-free media
and stained for 15 min in the dark. Imaging was done with Zoe Imager,
BioRad. The images were processed, and analysis of live–dead
staining and ROS staining was measured using the ImageJ tool, NIH,
USA.76 (link) Cell viability was calculated with
respect to untreated cells, and % ROS was calculated with respect
to nuclear counterstained cells.
× 104 cells/well in 12-well plates, differentiated,
and treated with rotenone (100 nM), Yashtimadhu choorna extract (200
μg/mL), 100 nM rotenone + Yashtimadhu choorna extract cotreatment
for 48 h. The untreated cells were taken as a control. Post-treatment,
the medium was aspirated, and the cells were washed with 1× phosphate-buffered
saline (PBS) and treated with 20 μg/mL of PI for dead cells
and 5 μg/mL of HOECHST as a counter nuclear stain in serum-free
media for 15 min.75 (link) For staining of the
intracellular ROS productions, the cells were stained with DCFDA post-treatment
with rotenone, Yashtimadhu alone, and Yashtimadhu choorna extract
cotreatment. The cells were washed with 1× PBS and stained with
25 μM of DCFDA and 5 μg/mL of HOECHST in serum-free media
and stained for 15 min in the dark. Imaging was done with Zoe Imager,
BioRad. The images were processed, and analysis of live–dead
staining and ROS staining was measured using the ImageJ tool, NIH,
USA.76 (link) Cell viability was calculated with
respect to untreated cells, and % ROS was calculated with respect
to nuclear counterstained cells.
5
Imaging C. elegans Nematodes
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Samples of control or exposed C. elegans nematodes were immobilized with 0.2 mM levamisole hydrochloride (Vetranal, Sigma–Aldrich), mounted on glass slides and imaged by light microscopy (Olympus CK40 or Leitz Fluovert FS) or fluorescence microscopy (Zeiss Axiovert M200). Nematodes on optical 96-well plates were also directly imaged using the Zoe imager (Bio-Rad Laboratories, Hercules, CA, USA). The intensity of fluorescence was measured from digital images using the ImageJ software (Wayne Rasband, NIH, USA).
6
Cytotoxicity Evaluation of CeO2 NPs
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The cytotoxic effect of the calcein-modified CeO2 NPs was assessed using a Live/Dead assay. This method was used to calculate the percentage ratio of the number of cells that died during incubation with the calcein-modified CeO2 NPs to their total number. Cell numbers were counted using fluorescence microscopy, and cells were labeled via staining with a combination of the fluorescent dyes Hoechst 33,342 (binds to DNA of all cells, excitation wavelength 350 nm, emission wavelength 460 nm) and propidium iodide (binds to DNA of dead cells, excitation wavelength 535 nm, emission wavelength 615 nm). After 24, 48, and 72 h of incubation, the culture medium was replaced with a mixture of Hoechst 33,342 fluorescent dyes and propidium iodide in Hanks’ buffer solution (PanEko, Moscow, Russia). After 15 min of incubation with dyes, the cells were washed three times with Hanks’ buffer solution and were further analyzed using a ZOE imager (Bio-Rad, USA). ImageJ software was used to count the number of cells. Three areas on the field were analyzed on three different microphotographs. Quantitative analysis was presented as mean ± SD.
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