Transwell inserts (membrane pore size, 8 μm; diameter, 6.4 mm; cat. no. 353097, FALCON®, Corning Inc., United States) were placed in 24-well plates. The cells were seeded in serum-free medium in upper chambers (150,000 cells per chamber for U87 and U87-TxR; 70,000 cells per chamber for C6 and RC6) covered with a Matrigel layer (500 ng/ml; cat. no. 356234, Basement Membrane Matrix, BD Biosciences, United States) and subsequently treated with 2 μM 5 and 8 μM 6 . The lower chambers were filled with appropriate medium supplemented with 10% fetal bovine serum as a chemoattractant. A negative control without 10% fetal bovine serum was also included in each experiment, as a measurement of spontaneous cell invasion. After 24 h, cells that invaded through the Matrigel and membrane were fixed in 4% paraformaldehyde. Cells remaining on the upper surface of the membrane were carefully removed with a cotton swab. Invading cells from the lower surface of the membranes were stained with Hoechst 33342. Membranes were carefully removed from inserts and placed on microscopic glass slides. Stained cells were counted under a Zeiss Axio Vert inverted fluorescent microscope at 10× magnification. Stained cells were counted in ImageJ software (1.48, Microsoft, Redmond, WA, United States).
Axiovert inverted fluorescent microscope
Manufactured by Zeiss
Sourced in Germany
The Axiovert inverted fluorescent microscope is a high-performance laboratory instrument designed for advanced microscopy applications. It features an inverted optical design, enabling the observation of samples from below. The microscope is equipped with fluorescence imaging capabilities, allowing researchers to visualize and analyze fluorescently labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
Transwell insert, by BD (2 mentions)
Basement membrane matrix, by BD (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Antifade medium, by Thermo Fisher Scientific (1 mentions)
Metamorph 4, by Universal Imaging (1 mentions)
Actinred 555 readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Oregon green 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
4 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Mowiol, by Merck Group (1 mentions)
8 protocols using axiovert inverted fluorescent microscope
1
Transwell Invasion Assay for Cancer Cells
2
Matrigel Invasion Assay for Glioma Cells
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In Matrigel invasion assay, Transwell inserts (membrane pore size, 8 μm; diameter, 6.4 mm; BD Biosciences Discovery Labware, USA) were placed in 24-well plates. 70000 RC6 cells were seeded in serum-free medium in the upper chambers covered with a layer of Matrigel Basement Membrane Matrix (5 mg/ml, BD Biosciences) and treated 24 h with 10 μM CoQ10, 250 μM TMZ, or their combination. Corresponding untreated cells were used as a positive control. The lower chambers were filled with DMEM medium supplemented with 10% FBS as chemoattractant. A negative control with serum-free medium in the lower chamber was also included in the experiment. After the incubation, cells that invaded through the Matrigel and its underlying membrane were fixed in 4% PFA and stained with Hoechst 33342 (1 : 1000). Cells (their nuclei) present at the lower surfaces of the membranes were counted under a Zeiss Axiovert inverted fluorescent microscope, at 10x magnification. The average number of cells in 30 independent fields per membrane was analyzed using ImageJ software. At least three independent experiments were performed.
3
Immunostaining and Fluorescence Microscopy Protocol
4
Quantifying Extracellular Matrix Degradation
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The potential of GBM cells to degrade the ECM, which corresponds to their migratory ability, was assessed by gelatin degradation assay. U87, U87-TxR and primary GBM cells were seeded in 6-well cell culture plates (50,000 cells per well) on glass coverslips coated with fluorescently labeled gelatin (Gelatin from Pig Skin, Oregon Green® 488 Conjugate, Thermo Fisher Scientific, MA, USA). U87 and U87-TxR cells were treated with 5 µM and 10 µM Si306 and pro-Si306, while primary GBM cultures were treated with 10 µM and 20 µM Si306 and pro-Si306. After 24 h, cells were fixed with 4% PFA in PBS and co-stained with Hoechst 33342 (Sigma-Aldrich Chemie Gmbh, Germany) to mark the nuclei and ActinRed™ 555 ReadyProbes™ Reagent (Thermo Fisher Scientific, MA, USA) to mark actin filaments. Cells and degraded area of gelatin were visualized at 20× magnification under a Zeiss Axiovert inverted fluorescent microscope. Size of the dark area under cells that corresponds to gelatin degradation was measured in ImageJ software and normalized to the number of nuclei in each image. At least 100 cells were analyzed per experiment.
5
Evaluating Matrix Degradation and Invasion
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The effect of Si306 and pro-Si306 on the ability of U87 and U87-TxR cells to degrade the matrix and invade through basement membrane was evaluated by Matrigel invasion assay using Transwell inserts (membrane pore size, 8 µm; diameter, 6.4 mm; BD Biosciences Discovery Labware, MA, USA). The cells were plated in a serum free medium in the upper chambers (150,000 cells/chamber) covered with a layer of 500 g/mL Matrigel (Corning Inc., NY, USA), a solubilized basement membrane preparation rich in ECM proteins laminin, collagen IV, heparin sulfate proteoglycans, entactin/nidogen and growth factors, and immediately treated with 5 µM Si306 and pro-Si306. The lower chambers were filled with MEM medium containing 10% FBS as a chemo-attractant. The control of spontaneous cell invasion in medium without 10% FBS was also included. After 24 h, cells that migrated through the membrane were fixed in 4% PFA in PBS, stained with Hoechst 33342 and counted under a Zeiss Axiovert inverted fluorescent microscope at 10× magnification. The average number of cells per membrane was analyzed by ImageJ software.
6
Visualizing Autophagolysosomes with Acridine Orange
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The autophagolysosomes were visualized by AO staining. After 48 h single treatments with 10 µM Si306 and pro-Si306 or co-treatments with 20 nM Baf A1, cells were washed in PBS and incubated for 15 min at 37 °C with 1 µM AO. Acridine orange accumulated in acidic compartments such as autophagolysosomes displays red fluorescence, while AO in cytoplasm and nuclei shows green fluorescence. After washing in PBS, cells were imaged live under Zeiss Axiovert inverted fluorescent microscope at 10× magnification using AxioVision 4.8 software.
7
Fractal Analysis of Microglial Morphology
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Black and white images of MHC-II+ microglia were obtained using a digital camera attached to a Zeiss axiovert inverted fluorescent microscope (Zeiss, Germany). The images were processed using ImageJ software (developed at the USA National Institutes of Health and available on the Internet).2 Under a 40X objective, cells were picked randomly in each selected area, and the binary overlay of a cell was created using thresholding procedure. In this method, all pixels with their gray level values higher than the threshold value were treated as belonging to the cell image. Other pixels were treated as background. For each cell the appropriate threshold value was defined manually at the level at which the binary overlay completely covered the whole cell body and processes. Finally, the binary silhouette of the whole cell was reduced to its one-pixel outline for estimation of the fractal dimensions with the FracLac 2.5 ImageJ plug-in (A. Karperien, Charles Stuart University).
8
Immunostaining of Phosphorylated H2A.X in GBM-6 Cells
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For immunostaining of phosphorylated H2A.X, GBM-6 cells were seeded (25,000 cells/chamber) in 4-well chamber slides (Nunc, Nalgene, Denmark) in 500 µL of appropriate medium and allowed to grow at 37 °C overnight. The cells were then treated for 72 h with 10 µM Si306 or pro-Si306. Next, the cells were washed in PBS, fixed in 4% paraformaldehyde for 15 min at room temperature and blocked in 0.5% BSA in PBS for 1 h. Anti-phospho-histone H2A.X antibody was applied at 1:1000 dilution in PBS/0.3% Triton X-100 and the cells were incubated overnight at 4 °C. After washing in PBS, fluorescent Alexa Fluor 488 anti-rabbit IgG (H + L) secondary antibody was applied at 1:1000 dilution in 0.5% BSA in PBS for 1 h at room temperature. To mark the nuclei, the cells were co-stained with hoechst 33342 and then mounted in Mowiol (Sigma-Aldrich Chemie GmbH). The cells were visualized under a Zeiss Axiovert inverted fluorescent microscope (Carl Zeiss, Jena, Germany) and imaged using AxioVision 4.8 software.
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