Cell growth was analyzed with MTS assay using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, WI) following the protocol. Cells were seeded in 96-well culture plates at a density of 5×103 cells/well. Cell viability was assessed by adding 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) to the culture medium and incubated for 4 h at 37 °C. The optical density was measured at 490 nm using Synergy 2 plate reader (Biotek, VT). Cell migration was measured by wound healing assays as described [18 (link)]. Briefly, cells were plated at a density of 1 x 105 cells per well in a 6-well plate were grown to confluence, at which time they were scratched with a micropipette tip. Phase-contrast images were captured with a Zeiss Axiovert 35 inverted microscope using AxioVision REL 4.6 imaging software (Zeiss) every 12 hr. The area of three representative wounds for each cell line was quantified at each time point using Image J software (NIH). Anchorage-independent growth soft agar assays were performed by seeding 4 x 103 cells in 0.3% agarose as described [19 (link)]. The colonies were quantified at day 14 from five fields in triplicate wells.
Axiovert 35
Manufactured by Zeiss
Sourced in Germany, United States
The Axiovert 35 is an inverted microscope designed for routine light microscopy applications. It features a sturdy, compact design and offers high-quality optics for bright-field and phase-contrast observation. The Axiovert 35 is suitable for various applications, including cell culture, tissue analysis, and educational purposes.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Gentamycine, by Merck Group (4 mentions)
Slide 6 0, by Ibidi (3 mentions)
Obis 532 ls laser, by Coherent Inc (2 mentions)
Labview, by National Instruments (2 mentions)
Water immersion objective, by Zeiss (2 mentions)
Fetal bovine serum solution fbs, by Merck Group (2 mentions)
Toto 3 iodide, by Thermo Fisher Scientific (2 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (2 mentions)
Verteporfin, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (2 mentions)
Signal averager software v 6, by Cambridge Electronic Design (2 mentions)
Coolpix 4500, by Nikon (2 mentions)
Synergy 2 plate reader, by Agilent Technologies (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Axiocam erc 5s, by Zeiss (1 mentions)
Matrigel mg, by Corning (1 mentions)
56 protocols using axiovert 35
1
Cell Proliferation, Migration, and Anchorage-Independent Growth Assays
2
Isolation and Cultivation of Marine Microalgae
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Individual cells were isolated from field samples using a glass Pasteur pipette drawn to a narrow filament with the aid of a Zeiss Axiovert 35 inverted microscope (Zeiss, Jena, Germany). Approximately 40 cells from each site were sequentially transferred through five drops of seawater taken from the original sample and sterilized by filtration before being transferred into individual wells of a 96-well plate containing 50% of f10K culture media [60 (link)] in sterile seawater. The plates were incubated at 26 °C with a 12:12 day/night photoperiod. Once the cells completed several division cycles, they were transferred to a 24-well culture plate containing f10K culture media and subsequently to 50 mL glass Erlenmeyer flasks for further growth. Cultures in the exponential growth phase were then pelleted using centrifugation from a known volume and cell density and stored at 20 °C until extraction.
3
3D Tumor Colony Proliferation Assay
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Cell proliferation was evaluated by average size and number of 3D tumor colonies under anchorage-independent growth conditions in 5 mg/mL Matrigel (MG; Corning Incorporated, NY, USA). Therefore, as explained above, a 96-well plate (Sarstedt AG & Co. KG, Nümbrecht, Germany) was coated with MG diluted in the appropriated culture medium. After solidifying at 37 °C, the upper layer of an MG cell suspension (38 cells/well, in a final volume of 53 µL cell MG suspension each well) was plated as described above. The plate was then incubated for 45 min at 37 °C and finally augmented with the adequate culture medium. The colony formation took 11 days of incubation at 37 °C and 5% CO2. A change in the medium was performed after 7 days. For the evaluation of colony number and surface area, images were captured using Zeiss Axiovert 35 and Zeiss Axiocam ERc 5s (Carl Zeiss, Jena, Germany) of colony number and surface area. ZEISS ZEN lite 3.5 (blue version) software (Carl Zeiss Microscopy, Oberkochen, Germany) was used to photograph the colonies at 10× magnification. The colony area was determined in µm2 by ImageJ 1.53 (NIH, NY, USA).
4
Immunofluorescence Analysis of Oxidative Stress
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Cells were grown on coverslips and after oxidative stress/recovery treatments they were washed twice in ice‐cold PBS. Next, the cells were fixed in 10% PBS‐buffered formaldehyde for 30 min. at room temperature, subsequently washed with PBS. To avoid formaldehyde autofluorescence, the coverslips were quenched with 50 mM ammonium chloride for 10 min. Cells were permeabilized with 0.1% Triton‐X 100 for 5 min. Non‐specific sites were blocked with 5% bovine serum albumin (Sigma‐Aldrich) in PBS for 5 min. and then the coverslips were incubated at room temperature with CTD110.6 monoclonal antibody for 30 min. at a dilution of 1:100 in 5% BSA/PBS. After rinsing three times with PBS, the samples were incubated with Alexa Fluor 594 goat antimouse IgM secondary antibody (1:200; Thermo Fisher Scientific) for 30 min. in dark. Nuclei were counterstained with Hoechst dye at a dilution of 1:5000 for 15 min. at room temperature. Finally, coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) mounting medium. Image acquisition was performed with a Zeiss Axiovert 35 (Carl Zeiss Microscopy GmbH, Jena, Germany) inverted fluorescent microscope with CellD (Olympus, Tokyo, Japan) software.
5
Single-Vesicle Docking and Fusion Visualization
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Experiments examining single-vesicle docking and fusion events were performed on a Zeiss Axiovert 35 fluorescence microscope (Carl Zeiss, Thornwood, NY), equipped with a ×63 water immersion objective (Zeiss; N.A. = 0.95) and a prism-based TIRF illumination. The light source was an OBIS 532 LS laser from Coherent Inc. (Santa Clara CA). Fluorescence was observed through a 610 nm band-pass filter (D610/60; Chroma, Battleboro, VT) by an electron multiplying CCD (DU-860E; Andor Technologies, Belfast UK). The prism-quartz interface was lubricated with glycerol to allow easy translocation of the sample cell on the microscope stage. The beam was totally internally reflected at an angle of 72° from the surface normal, resulting in an evanescent wave that decays exponentially with a characteristic penetration depth of ~100 nm. An elliptical area of 250 × 65 µm was illuminated. The laser intensity, shutter, and camera were controlled by a homemade program written in LabVIEW (National Instruments, Austin, TX).
6
Microscopy-based Cell Positioning Mapping
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Brightfield images with an upright light microscope (Zeiss Axioplan, ×10 objective, Carl Zeiss AG) related the electrically estimated cell positions to ground truth. For the CMOS MEA imaging, we reconstructed the whole microscopic image from individual image sections with the Fiji (Fiji is just ImageJ, version 1.54f, RRID: SCR_002285) plugin “Stitching” described in (Preibisch et al., 2009 (link)). Afterwards, we segmented the cell-covered area with the plugin “Trainable Weka Segmentation.” Brightfield images of 96-well plates were taken with an inverted microscope (Zeiss Axiovert 35, Carl Zeiss AG) with a ×10 objective.
7
Single Vesicle Docking and Fusion Imaging
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Experiments examining single-vesicle docking and fusion events were performed on a Zeiss Axiovert 35 fluorescence microscope (Carl Zeiss, Thornwood, NY), equipped with a 63x water immersion objective (Zeiss; N.A. = 0.95) and a prism-based TIRF illumination. The light source was an OBIS 532 LS laser from Coherent Inc. (Santa Clara CA). Fluorescence was observed through a 610 nm band pass filter (D610/60; Chroma, Battleboro, VT) by an electron multiplying CCD (DU-860E; Andor Technologies). The prism-quartz interface was lubricated with glycerol to allow easy translocation of the sample cell on the microscope stage. The beam was totally internally reflected at an angle of 72 o from the surface normal, resulting in an evanescent wave that decays exponentially with a characteristic penetration depth of ~100 nm. An elliptical area of 250 x 65 µm was illuminated. The laser intensity, shutter, and camera were controlled by a homemade program written in LabVIEW (National Instruments, Austin, TX).
8
Fura-FF-AM-based Ca2+ Measurement in Muscle Fibers
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Primary-cultured interosseous muscle fibres62 (link) were loaded with 5 µM of the ratiometric low-affinity Ca2+ indicator dye Fura-FF-AM in HEPES-buffered standard Krebs–Ringer solution for 30 min at room temperature, washed and equilibrated with Ringer solution containing 100 µM BTS for another 30 min to supress contractions. The bath solution was then replaced with Ca2+-free Ringer solution to eliminate the influence of external Ca2+. Target cells were superfused using Ca2+ releasing solution, i.e., Ca2+-free Ringer solution containing 500 µM of the RyR agonist 4-CmC to trigger Ca2+ release and 30 µM CPA to inhibit SERCA Ca2+ sequestration. A period of 20-s application of the Ca2+ releasing solution was always bracketed with 20-s superfusion with Ca2+-free Ringer solution (Fig. 3a ). For Ca2+ measurements, the ratio of fluorescence at 340 and 380-nm excitation was determined using the Zeiss Microscope Photometer System (FFP) based on an inverted microscope (Axiovert 35, Zeiss) equipped for epifluorescence (Fluar ×40/1.3 oil-immersion objective)63 (link).
9
Virus-like particle binding assay
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Cultured or ex vivo expanded cells were harvested and washed once with PBS. For each sample, 2 × 105 cells were resuspended in 50 µL PBS and incubated with 250 ng Atto-labeled MS2-VP1u (6 × 1010 particles) for 1 h at 37 °C. Cells were washed once with PBS, fixed with formaldehyde (2%), permeabilized with ethanol and analyzed by fluorescence microscopy (Axiovert 35, Carl Zeiss, Jena, Germany) or flow cytometry (BD LSR II, BD Biosciences, San Jose, CA, USA). Fluorescence images were processed with ImageJ software [27 (link)]; flow cytometry data was analyzed using the FlowJo software (FlowJo, Ashland, OR, USA).
10
Sperm Motility Characterization in Microfluidics
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