Three-dimensional growth assays were performed as described with the substitution of agarose to prevent cell adhesion [13] (link). Briefly, 2 × 104 cells were plated on top of 1.0% agarose in 6-well plates in media supplemented with cosmic calf serum (Complete, Thermofisher Scientific) or CSS (Midsci). Cells were left untreated or treated with DMSO (vehicle), Bay-11-7085 (Enzo Life Sciences, 1 μM), Casodex (Selleck Chemicals, 10 μM), or BMS-777607 (Selleck Chemicals, 5 μM) daily. After 10 days, images of spheres were taken using a Zeiss Axiovert S100TV inverted microscope (Carl Zeiss Microscopy), and spheres >25 μm in diameter were counted using ImageJ software. The 25-μm threshold was established based on the average sphere size obtained for the control cells. For 2D growth, 2.5 × 104 cells were plated on 12-well plates and counted every 24 hours. Myc-CaP Ctrl or Myc-CaP Ron OE cells were grown in 2D and treated with DMSO (vehicle), LiCl (Sigma, 10 mM, 4 hours), or Bay-11-7085 (Bay 11, 5 μM, 4 hours) when cells were ~70% confluent prior to fractionation.
Axiovert s100 tv
Manufactured by Zeiss
Sourced in Germany, United States, United Kingdom
The Axiovert S100 TV is a high-quality inverted microscope designed for various laboratory applications. It features a sturdy, stable construction and provides essential optical performance for routine microscopy tasks.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfap, by Novus Biologicals (2 mentions)
Bay 11 7085, by Merck Group (1 mentions)
Bms 777607, by Selleck Chemicals (1 mentions)
Bay 11 7085, by Enzo Life Sciences (1 mentions)
1.3 na oil immersion objective, by Zeiss (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Anti nestin, by Merck Group (1 mentions)
Axiovert 100 inverted microscope, by Zeiss (1 mentions)
Dab staining, by Vector Laboratories (1 mentions)
Anti ki67 clone sp6, by Lab Vision (1 mentions)
Filter cube, by Chroma Technology (1 mentions)
Hxp 120, by Zeiss (1 mentions)
Visiview software, by Visitron (1 mentions)
Nanoscope software, by Digital Instruments (1 mentions)
Metamorph imaging software, by Molecular Devices (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
M165 fc, by Leica (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Orca flash 4.0 cmos camera, by Hamamatsu Photonics (1 mentions)
15 protocols using axiovert s100 tv
1
Three-dimensional Growth Assay for Spheroid Analysis
2
3D Growth Assay for Sphere Formation
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Three‐dimensional growth assays were performed as described previously.11 , 14 Briefly, 2 × 104 cells were plated on top of 1.0% agarose in six‐well plates in media supplemented with cosmic calf serum (Complete; Thermofisher Scientific) or charcoal‐stripped serum (CSS, Midsci). Cells were left untreated, treated with PBS (vehicle), Gas6 (R&D, 1 nM daily), or macrophage conditioned media (CM) (R&D, 1:10 every other day) daily. After 10 days, images of spheres were taken using a Zeiss Axiovert S100TV inverted microscope (Carl Zeiss Microscopy), and spheres >25 µm in diameter were counted using ImageJ software. The 25 μm threshold was established based on the average sphere size obtained for the control cells.
3
3D Growth Assay for Sphere Formation
4
3D Co-culture Spheroid Assay
5
Monitoring Cytosolic Calcium Changes in Islets
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Cytosolic [Ca2+] changes were monitored as ratiometric measurements of Fura-2 fluorescence. Isolated mouse islets were cultured on glass coverslips treated with poly-L -lysine (Sigma-Aldrich) and placed in a thermostatic chamber (Harvard Apparatus, Holliston, MA) before incubation with 2 µM Fura-2/acetoxymethyl ester (AM) for 60 min. After washing, Fura-2 fluorescence of a single islet was imaged with alternate 340/380 nm excitation and 510 nm emission using an Axiovert S100 TV through a 40 × 1.3 NA oil immersion objective (Carl Zeiss GmbH, Jena, Germany) as described [38] (link). After the 22.8 mM glucose stimulation, 1 μM thapsigargin was added to evaluate the contribution of endoplasmic reticulum to cytosolic [Ca2+] changes. Data obtained from islets of at least 3 mice were presented both as 340/380 ratio and normalized to cytosolic [Ca2+] at low glucose.
6
Quantifying Axon Retraction in Retinal Cultures
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To examine axon retraction, a Zeiss inverted light microscope (Axiovert S100TV; Carl Zeiss), equipped with a motorized stage (Assy Stage 25; Cell Robotics, Inc., Albuquerque, NM, USA) and a 40×, 0.75 N.A. objective, was used to view retinal cultures. Rod photoreceptors were identified by morphology (cell shape and presence of an ellipsoid and axon terminal). Rod cells with axon terminals (20–23 per dish) were selected by brightfield microscopy within the first hour after cell plating by viewing the culture at an arbitrary location and then systematically scanning in rows. At 7 hours, the same cells were located again. Their images were captured with a charge-coupled device camera (XC 75CE; Sony, Japan).
Axon length was measured with ImageJ. Reduction in length, indicating retraction, was the difference between the axonal lengths at the 1st (L1) and 7th (L7) hour of the same cell. The percent decrease in length was calculated with the following formula: (L1-L7) ÷ L1 × 100%.
Axon length was measured with ImageJ. Reduction in length, indicating retraction, was the difference between the axonal lengths at the 1st (L1) and 7th (L7) hour of the same cell. The percent decrease in length was calculated with the following formula: (L1-L7) ÷ L1 × 100%.
7
Immunohistochemical Analysis of Brain Tissue
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For immunohistochemical analysis, tissue sections were de-paraffinized, rehydrated and subjected to high temperature antigen retrieval in 10 mM citrate buffer (pH 6.0). The following primary antibodies were used: rabbit monoclonal anti-Ki67 (clone SP6, Lab Vision, 1:100) rabbit polyclonal anti-GFAP (Novus Biologicals, 1:100), anti-nestin (Millipore, 1:100). These primary antibodies were applied for 2 h in blocking buffer (2.5% BSA, 5% goat serum, 0.3% Triton X-100 in PBS), followed by species-appropriate secondary Alexa Fluor 488 dye conjugated antibodies (Amersham) or Vectastain ABC kit and DAB reagents (Vector Laboratories). Fluorescence antibody-labeled slides were mounted in DAKO fluorescent mounting medium containing 1 μg ml−1 Hoechst counterstain. HRP-conjugated secondary antibodies were visualized by DAB staining (Vector Laboratories). Apoptotic cells were detected with ApopTag Detection kit (Chemicon International). Images were obtained with an Axiovert S100 TV inverted fluorescence microscope (Zeiss) and Open Lab 3.5.1 software, or with an Axiovert 100 inverted microscope (Zeiss) equipped with a Hamamatsu Orca digital camera.
8
Immunofluorescence Analysis of Ki67 and GFAP
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NPGs were centrifuged with CytoSpin (Eppendorf) to cause them to adhere onto slides and fixed in 4% paraformaldehyde. Fixed cells were blocked with 5% BSA–0.1% Triton and then incubated with rabbit monoclonal anti-Ki67 (clone SP6, Lab Vision, 1:100) and rabbit polyclonal anti-GFAP (Novus Biologicals, 1:500) overnight and counterstained with Hoechst. Images were collected with an Axiovert S100 TV inverted fluorescence microscope (Zeiss) and Open Lab 3.5.1 software.
9
Live-Cell Imaging using Inverted Microscope
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Light microscopy was performed on an inverted Axiovert S100TV (Carl Zeiss) epifluorescence microscope equipped with electronic shutters (Uniblitz Electronic 35 mm including driver Model VMMD-1, BFI Optilas), a filter wheel (LUDL Electronic products LTD), filter cubes (Chroma Technology Corp. Rockingham), epifluorescence illumination (light source HXP 120, Zeiss), and a tungsten lamp (Osram, HLX64625, FCR 12 V, 100 W) for phase contrast optics. Cells were observed in an open chamber (Warner Instruments) with a heater controller (Model TC-324 B, SN 1176) at 37 °C. Ham’s F12 medium containing all growth supplements was used for imaging. Live cell imaging was performed using 63x/1.4-NA or 100x/1.4-NA plan apochromatic objectives. Images were acquired with a back-illuminated, cooled, charge-coupled-device (CCD) camera (CoolSnap HQ2, Photometrics) driven by VisiView software (Visitron Systems).
10
Measuring Cellular Elasticity via AFM
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Single dissociated cells were put on a glass bottom dish in 1×MBS-H. They were compressed using a Bioscope Atomic force microscope (AFM) (Veeco) mounted on an inverted optical microscope (Axiovert S100 TV; Zeiss) with a Silicon Nitride cantilever with a nominal spring constant of K = 0.01 (MLCT; Veeco) mounted with a 10 µm polystyrene bead (Polysciences). Force-distance (FD) curves were obtained with the Nanoscope software (Digital Instruments). FD curves were plotted as the deflection of the cantilever as it was lowered in the z-axis to make contact with, and compress single cells. The elastic modulus was calculated by fitting the first 200 nm of each FD curve using a Hertzian compression model for a spherical indenter compressing a spherical substrate. Code kindly provided by X.Y. Chau and P. Grütter, McGill University, available at http://spm.physics.mcgill.ca/research-projects/afm-in-fluids/mechanical-properties-of-neurons . Multiple approaches were taken for each cell and the average elastic modulus for each cell was used as a single data point. Statistical analysis was done using a one-way ANOVA followed by Tukey’s HSD post hoc test for individual comparisons.
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