SC migration was assessed using a transwell-based assay as previously described (Parrinello et al., 2010 (link)). In brief, SCs were seeded onto the upper chamber of a transwell insert with 8 μm pores (Costar, Cambridge, MA) at a cell density of 1 × 106 cells/mL in DMEM. To evaluate SC migration, the cells were allowed to migrate at 37°C in 5% CO2 for 24 h, after which the transwell insert was removed and the upper surface of its insert membrane cleaned with a cotton swab. The cells on the bottom surface of the insert membrane were next stained with 0.1% crystal violet for 30 min at room temperature and subsequently imaged and counted under a DMR inverted microscope (Leica).
Dmr inverted microscope
Manufactured by Leica
Sourced in Germany, China
The Leica DMR inverted microscope is a versatile laboratory instrument designed for a wide range of microscopy applications. It features a robust and ergonomic construction, providing a stable platform for high-quality optical performance. The DMR is capable of a variety of imaging techniques, including brightfield, phase contrast, and fluorescence microscopy, enabling researchers to study a diverse range of samples with precision and accuracy.
Automatically generated - may contain errors
Lab products found in correlation
6.5 mm transwell chambers with 8 μm pores, by Corning (6 mentions)
Fibronectin, by Merck Group (3 mentions)
Crystal violet, by Beyotime (3 mentions)
Tcs sp2 laser scanning spectral system, by Leica (2 mentions)
6.5 mm transwell chambers with 8 mm pores, by Corning (2 mentions)
Image pro plus, by Media Cybernetics (2 mentions)
Matrigel, by BD (2 mentions)
Transwell chamber, by Corning (2 mentions)
6.5 mm transwell chamber, by Corning (2 mentions)
Transwell insert, by Corning (1 mentions)
Mowiol, by Merck Group (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Transwell cell culture chamber, by Corning (1 mentions)
Crystal violet, by Agilent Technologies (1 mentions)
Cell light edu dna cell proliferation kit, by RiboBio (1 mentions)
Recombinant rat il 4, by Thermo Fisher Scientific (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
6.5 mm transwell with 8 μm pores, by Corning (1 mentions)
6.5 mm transwell chamber with 8 μm pores, by Corning (1 mentions)
41 protocols using dmr inverted microscope
1
Schwann Cell Migration Assay
2
Transwell Assay for Schwann Cell Migration
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Schwann cells were resuspended in DMEM only medium and seeded at a density of 3 × 105 cells/mL onto the top chamber of 6.5 mm transwell chamber with 8-µm pores (Costar, Cambridge, MA, USA). Complete medium containing 10% fetal bovine serum was added to the bottom chamber of the transwell. Schwann cells were incubated for additional 24 hours. The upper surface of the top chamber of transwell was cleaned with a cotton swab and the bottom surface of the top chamber was stained with crystal violet. Images were taken using DMR inverted microscope (Leica). Crystal violet labeled migrated Schwann cells were washed out by 33% acetic acid and the absorbance of crystal violet was measured at 570 nm (Bio-Tek). Cell migration abilities were determined by the absorbance of crystal violet and normalized to the non-targeting control.
3
Quantifying Cell Migration via Transwell Assay
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Cell migration was assayed using Transwell inserts (Corning Inc, Corning, NY, USA) (Mantuano et al., 2008). The membrane of each insert was coated with fibronectin (Sigma). Schwann cells were planted in the top chamber with Dulbecco’s modified Eagle’s medium. The lower chambers contained complete medium. After 24 hours, the migrated Schwann cells were fixed with methanol and stained with crystal violet solution. The non-migrated cells in the upper chamber were wiped with cotton swabs. Migrated cells were imaged and tallied using a DMR inverted microscope (Leica, Mannheim, Germany). The migrated cell numbers were calculated, taking the average number of migrated cells in control group as 100%. The cell migration rate of the p-GV230-Claudin-15 group was obtained by dividing it with the average number of negative control or pGV230 group. The results were exhibited as fold changes.
4
Subcellular Localization of ABCB4 Variants
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ABCB4-wt- and ABCB4-ΔQNL-expressing HepG2 cells were fixed with methanol/acetone (4:1, v/v) at -20°C. Incubations with primary and secondary antibodies were performed as previously described [8 (link)]. Confocal imaging was acquired with a Leica TCS SP2 Laser Scanning Spectral system attached to a Leica DMR inverted microscope. Optical sections were recorded with a 63/1.4 oil immersion objective. Laser scanning confocal images were collected, and analyzed using the on-line “Scan Ware” software. Images were processed using ImageJ 1.41 and Photoshop softwares. Figure compilation was accomplished using Adobe Photoshop 5.5 and Adobe Illustrator 10.
5
Immunofluorescence Microscopy of Cultured Cells
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Cells were cultured on glass cover-slips in 24 well plates as described in (Illien et al. 2012) .
The cells were washed with PBS, and fixed with paraformaldehyde (3.7% ) in PBS for 15 minutes at room temperature (Ayala et al. 1989 ). Then they were washed three times with PBS, NH4Cl 50 mM and post-fixed and permeabilized with cold methanol (-20°C) for 1 minute and washed as before. Fixed cells were saturated with PBS, NH4Cl 50 mM, BSA 1%
for 20 minutes at room temperature. The first antibodies were added at the right dilution during 1.5 hours at room temperature followed by three washes in saturation solution. The secondary antibodies were incubated and washed as the first antibodies. Finally, the coverslips were dried and mounted in Mowiol (Sigma-Aldrich). The fluorescence confocal images were acquired with a TCS SP2 laser-scanning spectral system (Leica, Wetzlar, Germany) attached to a Leica DMR inverted microscope at the specific excitation wavelengths and appropriate emission channels for each fluorophore. Optical sections were recorded with a 63/1.4 or 100/1.4 immersion objectives.
The cells were washed with PBS, and fixed with paraformaldehyde (3.7% ) in PBS for 15 minutes at room temperature (Ayala et al. 1989 ). Then they were washed three times with PBS, NH4Cl 50 mM and post-fixed and permeabilized with cold methanol (-20°C) for 1 minute and washed as before. Fixed cells were saturated with PBS, NH4Cl 50 mM, BSA 1%
for 20 minutes at room temperature. The first antibodies were added at the right dilution during 1.5 hours at room temperature followed by three washes in saturation solution. The secondary antibodies were incubated and washed as the first antibodies. Finally, the coverslips were dried and mounted in Mowiol (Sigma-Aldrich). The fluorescence confocal images were acquired with a TCS SP2 laser-scanning spectral system (Leica, Wetzlar, Germany) attached to a Leica DMR inverted microscope at the specific excitation wavelengths and appropriate emission channels for each fluorophore. Optical sections were recorded with a 63/1.4 or 100/1.4 immersion objectives.
6
Wound Healing Assay of Stem Cells
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A wound-healing assay was performed as described previously (18, 23) with modifications. SCs were seeded onto the bottom chamber of a 24-well plate, incubated until confluent, and transfected with synthetic miR-sc3, an miR-sc3 inhibitor, or nontargeting negative controls for 24 h. Transfected SCs were starved in DMEM supplemented with 0.5% FBS (Invitrogen) and 0.15 μg/ml mitomycin C (Sigma-Aldrich) for 12 h and then wounded by a sterile pipette tip to form a linear scratch. The wounded cell monolayers were imaged under a DMR inverted microscope (Leica) at 0 and 12 h after wounding. The relative wound closure at 12 h compared with 0 h was measured using Wimscratch Quantitative Wound Healing Image Analysis (Wimasis GmbH, Munich, Germany).
7
Quantifying Schwann Cell Migration
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Cell migration assays were performed in Transwell cell-culture chambers (Costar, Cambridge, MA, USA) as described previously (Mantuano et al., 2008). The lower surface of each membrane was coated with fibronectin. Schwann cells were transferred to the top chambers of each transwell and allowed to migrate. The complete medium was injected into the lower chambers. The non-migrated cells were cleaned with a cotton swab at the indicated time point. Migrated cells were fixed with methanol and then stained with crystal violet solution. Cell migration was defined by images of randomly selected fields obtained on fluorescence. Cell migration imaging and counting occurred using a DMR inverted microscope (Leica Microsystems).
8
Transwell Assay for Schwann Cell Migration
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The migration rate of Schwann cells was measured using a transwell-based assay, as previously described (Li et al., 2015). Briefly, the bottom surfaces of 6.5-mm transwell chambers with 8 mm pores (Costar, Cambridge, MA, USA) were coated with fibronectin. Schwann cells in Dulbecco’s modified Eagle’s medium were seeded onto the upper chamber, and the bottom chamber was filled with complete culture medium. After 24 hours of culture, Schwann cells remaining on the upper surface were removed by a cotton swab. Schwann cells that migrated to the lower surface were stained by crystal violet (Beyotime, Shanghai, China). Migration images were obtained using a DMR inverted microscope (Leica Microsystems). Next, 33% acetic acid was used to wash crystal violet labeled migrated Schwann cells, and absorbance of crystal violet (Bio-tek, Winooski, VT, USA) was measured at 570 nm to determine cell migration ability.
9
Quantifying Microglia Proliferation In Vitro
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Highly aggressive proliferating immortalized microglia cells were cultured in the 96-well plates, and proliferation was detected by Cell-Light EdU DNA cell proliferation kit (RiboBio, Guangzhou, China) according to the instructions of the manufacturer. Briefly, cells were incubated with a culture medium containing 50 μM EdU for 2 h, followed by fixation in 4% formaldehyde for 30 min. Then, they were decolorized and incubated with 2 mg/ml glycine for 5 min and permeabilizated with 0.5% Triton X-100 for 10 min. After extensive washing with PBS, the cells were incubated with Apollo for 30 min and then treated with Hoechst for 30 min. Images were acquired with DMR inverted microscope (Leica Microsystems). Proliferation index = (number of EdU-positive cells/number of total cells) × 100%.
10
Transwell Migration Assay for M1/M2 Microglia
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For migration assays, 6.5 mm Transwell chambers with 8 µm pores (Corning Incorporated, Corning, NY, USA) were used as previously described (Zhou et al., 2018). Briefly, 100 µL of BV2 cells (6 × 105 cells/mL, FuHeng Biology, Shanghai, China, Cat# FH0355) were added into the top chamber of the Transwell following treatment with 20 ng/mL recombinant rat IL-4 (for induction of M2 phenotype; PeproTech, Cranbury, NJ, USA, Cat# 400-04) or 0.5 µg/mL lipopolysaccharide (LPS; for induction of M1 phenotype; MCE, Cat# HY-D1056) for 24 hours. Next, 600 µL of astrocytes (1 × 105 cells/mL) were placed into the lower chamber after CCL2 siRNA interference for 24 hours followed by stimulation with 1 µg/mL recombinant MIF. Cells were cultured for 24 hours at 37°C in 5% CO2. The upper surface of each membrane was cleaned with a cotton swab, and cells that adhered to the bottom surface were stained with 0.1% crystal violet. The migrated cells were imaged by a DMR inverted microscope (Leica Microsystems, Wetzlar, Germany). The number of migrated cells was counted using ImageJ.
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