Two thousand HLECs treated with TNF-α, MALAT1 siRNA, or negative control siRNA were inoculated on the upper surface of a 6.5 mm transwell chamber (BD Biosciences, San Jose, CA, USA), while the lower surface of the chamber was filled with 600 μL of medium containing 10% FBS. After 24 h of incubation, the chamber was fixed in 4% formaldehyde for 30 min and then stained with crystal violet for 15 min. The penetrating cells were observed under an Olympus CKX53 inverted fluorescence microscope and counted in three random fields.
Ckx53 inverted fluorescence microscope
Manufactured by Olympus
Sourced in Japan
The CKX53 inverted fluorescence microscope is a laboratory instrument designed for observation and analysis of biological samples. It features an inverted optical system that enables the examination of cells and tissues from the underside. The CKX53 is capable of fluorescence imaging, allowing for the visualization of labeled cellular components or structures.
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Lab products found in correlation
Lsm 900 confocal microscope, by Zeiss (3 mentions)
Primary htm cells, by ScienCell (1 mentions)
Cell medium tmcm, by ScienCell (1 mentions)
Tunel assay, by Beyotime (1 mentions)
Microplate reader, by Tecan (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Dp72 camera, by Olympus (1 mentions)
Imagexpress micro confocal high content imaging system, by Molecular Devices (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Beyotime (1 mentions)
Typhoon5 biomolecular imager, by GE Healthcare (1 mentions)
13 protocols using ckx53 inverted fluorescence microscope
1
Transwell Migration Assay of HLECs
2
Transduction of Primary Human Trabecular Meshwork Cells
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Primary HTM cells (ScienCell, Carlsbad, CA, USA) were cultured in TM Cell Medium (TMCM; ScienCell) at 37°C in an atmosphere of 5% CO2, as previously described.10 (link),11 (link),61 (link) Cells of passages 3 to 4 were used in the experiments. For western blot experiments, HTM cells (1 × 106 cells per 25-cm2 culture flask) were transduced with scAAV2-C3 (MOIs = 1.5 × 104 and 7.5 × 103) or scAAV2-EGFP (MOI = 1.5 × 104). For actin labeling, HTM cells were cultured to 100% confluence (4 × 105 cells per well in a 24-well plate) on coverslips, and then the cells were transduced with scAAV2-C3 or scAAV2-EGFP (MOI = 1.25 × 104). Images of morphology and EGFP expression were taken using an Olympus (Tokyo, Japan) CKX53 inverted fluorescence microscope.
3
Apoptosis Analysis of AGS Cells
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The apoptosis of AGS cells transfected with miR-183-5p.1 oligonucleotides was analyzed using a Hoechst 33258 detection kit (Beyotime Institute of Biotechnology; cat. no. C1011) and TUNEL assay (Beyotime Institute of Biotechnology; cat. no. C1089) according to the manufacturer's protocols. After transfection, cells were washed with PBS twice and incubated with Hoechst 33258 reagent (50 µl) for 60 min at 37°C in the dark, and then washed with PBS twice. After this, cells were stained with TUNEL solution (50 µl) for 60 min at 37°C in the dark and washed with PBS twice. Images were captured and the cells were counted using an Olympus CKX53 inverted fluorescence microscope (magnification, ×200). The immunostained sections were evaluated by two independent pathologists.
4
Immunofluorescence Staining of Stem Cells
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Cells were fixed with pre‐cooled methanol at 0–5°C for 20 min. After washing off the fixative, the cells were blocked with 3% BSA for 30 min at 15–25°C. The blocking solution was discarded, and cells were incubated with SOX‐2 (1:400) or CD133 (1:500) primary antibody. After washing away the unbound primary antibody, anti‐mouse IgG (H + L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) (1:500) and anti‐rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor® 594 Conjugate) (1:400) to incubate cells for 2 h, and then 0.5 μg/ml DAPI (with antifluorescence quencher) was added for 10 min. Cells were imaged under a CKX53 inverted fluorescence microscope (Olympus, Japan).
5
Mitochondrial Membrane Potential Assay
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HeLa, Jurkat, and HCT116 cells were used
for the assay, and the
assay was performed as described previously.40 (link) The treated cells were allowed to incubate for 24 h and subjected
for MMP assay as per th emanufacturer’s protocol (MAK159, Sigma-Aldrich).
Spectrophotometric absorbance of the plate was recorded at (λex = 490/λem = 525 nm) and (λex = 540/λem
= 590 nm) for ratio analysis using Tecan Microplate Reader (Tecan
Instruments, Switzerland). Cells were centrifuged at 800 rpm and imaged
using an Olympus CKX53 inverted fluorescence microscope.
for the assay, and the
assay was performed as described previously.40 (link) The treated cells were allowed to incubate for 24 h and subjected
for MMP assay as per th emanufacturer’s protocol (MAK159, Sigma-Aldrich).
Spectrophotometric absorbance of the plate was recorded at (λex = 490/λem = 525 nm) and (λex = 540/λem
= 590 nm) for ratio analysis using Tecan Microplate Reader (Tecan
Instruments, Switzerland). Cells were centrifuged at 800 rpm and imaged
using an Olympus CKX53 inverted fluorescence microscope.
6
Cell Morphology Analysis on PDMS Gels
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TDCs were seeded at a density of 104 cell/well in 24-well plates with the PDMS gels representing physiological, stiff and soft stiffness and incubated in DMEM/F12 with 5% FBS for 24 h. Cells were fixed in 4% paraformaldehyde for 20 min, and permeabilised with 0.5% triton-X overnight. For actin cytoskeleton staining, cells were incubated overnight with Alexa-Fluor™ 594 Phalloidin (Invitrogen™, Thermo Fisher Scientific), following the manufacturer protocol. Cells were then incubated for 3 h with DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride, Sigma Aldrich) for nuclear staining. Images of the fixed and stained cells were captured using Olympus CKX53 inverted fluorescence microscope using Olympus DP-72 camera. Cell periphery was manually traced from the images to calculate cell area, aspect ratio (the ratio between the major axis and the minor axis of the cells, AR) and circularity (defined as . More than 100 cells were captured per experimental group, taken from 10 images across 5 replicate wells in 3 independent biological experiments. Quantitative analysis of cell morphology was carried out using the NIH-ImageJ software (http://rsb.info.nih.gov/ij/ ).
7
Immunohistochemical Analysis of NLRP3, Caspase-1, and TLR4 in BV2 Cells and Rat Brain Tissue
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Briefly, BV2 cells were collected and fixed with 4% paraformaldehyde, embedded in dehydrated paraffin, and cut into 4 μm thick sections for IHC staining. The brain tissue of rats was prepared into paraffin sections. Subsequently, sections were dewaxed with xylene and subjected to antigenic repair with ethylenediamine tetraacetic acid (EDTA; pH =8.0) antigenic repair solution. After blocking endogenous peroxidases with 3% hydrogen peroxide for 30 min, sections were incubated with primary antibodies against NLRP3 (1:300), caspase-1 (1:500), and TLR4 (1:250) overnight at 4 °C. The following day, sections were incubated with an appropriate secondary antibody (1:350) for 50 min at room temperature. Next, sections were stained with a 3,3’-diaminobenzidine (DAB) colorimetric kit and hematoxylin. After dehydration and drying, neutral gum was applied to seal sections. Images were collected using a CKX53 inverted fluorescence microscope (Olympus, Tokyo, Japan), and percentages of positive staining (brown) were measured with ImageJ.
8
Nanog Expression Profiling in Reprogrammed Cells
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Cells reprogrammed with Oct4-SK were washed three times with PBS (1×) and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were permeabilised by incubation with 0.2% Triton X-100 for 15 min in 1× PBS at room temperature. The permeabilised cells were washed twice with 1× PBS and blocked with 5% BSA in 1× PBS for 1 h. Cells were incubated with primary Nanog antibody (Novus; #NB100–58842, 1:500, Supplementary Table 11 ) at 4 °C overnight. Cells were washed three times with 1× PBS and incubated with Alexa Fluor 594 Donkey anti-Rabbit IgG (H + L) secondary antibody (Invitrogen; #A21207, 1:1000) in the dark at room temperature for 1 h. Cells were washed three times with 1× PBS and further stained with 1× DAPI (Thermo Fisher Scientific; #R37606) and imaged with an Olympus CKX53 inverted fluorescence microscope. Whole-well scans were obtained by an ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices).
9
EdU Proliferation Assay in Cells
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The ×2 EdU working solution was made by diluting 10 μM of EdU (Beyotime) with MEM medium (1:500) and warmed to 37°C. An equal volume of ×2 EdU working solution was added to each well and the plate was incubated at room temperature for 2 h, washed with PBS and fixed with 4% paraformaldehyde for 15 min. The plates were then washed twice with PBS containing 3% bovine serum albumin (BSA) to remove the permeabilization solution. Click-iT ® cell buffer additive solution (Thermo Fisher Scientific) was added and each plate was incubated at room temperature for 30 min in the dark. Hoechst 33342 reaction solution was added, and the plates were incubated at room temperature for 10 min in the dark. The plates were washed 3 times before being observed under CKX53 inverted fluorescence microscope (Olympus, Tokyo, Japan). The red fluorescence was counted as proliferating cells, and the blue fluorescence showed nuclei. We chose 3 random ×200 fields to count the proliferating cells with nuclei. The cell proliferation rate = the number of proliferating cells/total cell number × 100%.
10
Apoptosis Induction by TJ08 Treatment
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Jurkat
cells were seeded at 1 × 105 cells/mL in 6-well dishes
and allowed to grow overnight. Cells were treated with different concentrations
ofTJ08 10 and 20 μM and allowed to incubate for
48 h. Cells were harvested, washed with cold PBS, and subjected for
Annexin V FITC/PI double staining assay. Samples were resuspended
in 1× binding buffer and incubated with Annexin V FITC and PI
according to the manufacturer’s protocol (Roche) and imaged
using Olympus CKX53 inverted fluorescence microscope.
cells were seeded at 1 × 105 cells/mL in 6-well dishes
and allowed to grow overnight. Cells were treated with different concentrations
of
48 h. Cells were harvested, washed with cold PBS, and subjected for
Annexin V FITC/PI double staining assay. Samples were resuspended
in 1× binding buffer and incubated with Annexin V FITC and PI
according to the manufacturer’s protocol (Roche) and imaged
using Olympus CKX53 inverted fluorescence microscope.
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