In the migration assay, OSCC cells were mixed with cultured medium containing 0.5% FBS and seeded into the upper chambers of the inserts with an 8-μm pore size of polyethylene terephthalate membrane (Millipore). The inserts were placed in 24-well plates containing complete medium with a series concentration of CXB (0, 25, 50 and 100 μM) in lower wells, respectively. The cell number ranged from 1 × 105 to 2 × 106 cells to evaluate the diversity characteristics of the migration or invasion activity among OSCC cells. After 24 h of treatment, the inserts were fixed in 4% paraformaldehyde. The top-chamber cells were removed by swabbing and migrated cells were stained with 0.1% crystal violet (w/v) in 20% methanol (v/v). The migration ability was observed and captured under a microscope (DM IL, Leica Microsystems). The invasiveness of treated OSCC cells (0, 25, 50 and 100 μM) was determined using a BioCoat Matrigel Invasion Chamber (Corning) set according to the Cell Invasion Assay protocol (http://csmedia2.corning.com/LifeSciences/media/pdf/protocol_DL_031_Cell_Invasion_Assay.pdf ).
Dm il
Manufactured by Leica
Sourced in Germany, Italy, United States, Japan
The Leica DM IL is a high-performance inverted microscope designed for laboratory applications. It features a sturdy and ergonomic construction, providing a stable platform for precise observations and analyses. The microscope's core function is to enable clear and detailed visualization of specimens, supporting a wide range of research and diagnostic activities.
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Lab products found in correlation
Dfc300fx, by Leica (5 mentions)
Automated cell counter, by Thermo Fisher Scientific (3 mentions)
Biocoat matrigel invasion chamber, by Corning (2 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
24 well insert, by Corning (2 mentions)
Dfc340fx, by Leica (2 mentions)
Live dead viability assay kit, by Thermo Fisher Scientific (2 mentions)
Polyethylene terephthalate membrane, by Merck Group (1 mentions)
Las ez software, by Leica (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
Tlrl ppglps, by InvivoGen (1 mentions)
Mscbm cd, by Lonza (1 mentions)
Mscgm cd, by Lonza (1 mentions)
Mscgm cd medium, by Lonza (1 mentions)
Leukocyte acid phosphatase kit, by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
86 protocols using dm il
1
Investigating OSCC Cell Migration and Invasion
2
In Vitro Wound Healing Analysis
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A wound-healing analysis was executed to assess the percentage of wound closure after the treatments [20 (link)].
Human PDLSCs were cultured in a concentration of 3 × 105 cells/dish. Twenty four hours before the treatment, cells were maintained in DMEM (Lonza) for cellular starvation. A sterile pipette with 10 µL tip was used to wound the cell monolayer. At defined time points after wounding (2 h, 6 h, 24 h and 48 h), an inverted light microscope (DMIL, Leica Microsystem, Milan, Italy) was used to analyze the rate of wound closure, calculating the migrated distance/total wound distance using the LEICA LAS/EZ software (version 3.4, Leica Microsystem, Milan, Italy).
Human PDLSCs were cultured in a concentration of 3 × 105 cells/dish. Twenty four hours before the treatment, cells were maintained in DMEM (Lonza) for cellular starvation. A sterile pipette with 10 µL tip was used to wound the cell monolayer. At defined time points after wounding (2 h, 6 h, 24 h and 48 h), an inverted light microscope (DMIL, Leica Microsystem, Milan, Italy) was used to analyze the rate of wound closure, calculating the migrated distance/total wound distance using the LEICA LAS/EZ software (version 3.4, Leica Microsystem, Milan, Italy).
3
Invasion Assay of SKOV-3 Cells
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The invasion behavior of the cells was determined using 24-well BD BioCoat™ Matrigel™ Invasion Chambers (8-μm pore size polycarbonate filters; BD Biosciences). Briefly, after the cells were treated for the indicated condition, 5 × 104 SKOV-3 cells in 200 μl of serum-free Dulbecco’s modified Eagle’s medium (DMEM) were plated onto the upper chambers, while complete medium containing 10% FBS was added to the lower chamber. After incubating the invasion chambers for 48 h at 37°C with 5% CO2, the noninvaded cells were removed with a cotton swab. The invaded cells were fixed in 100% methanol and then stained with crystal violet solution and counted under a microscope (DMI L; Leica Microsystems). The data are presented as the average number of cells attached to the bottom surface from five randomly chosen fields.
4
Isolated hPDLSCs Treated with LPS and LYO
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The following experiments were performed using the isolated hPDLSCs at passage 2. Here, we reported the experimental groups:
Untreated hPDLSCs, used as negative control (CTR);
hPDLSCs treated with lyophilized conditioned medium resuspended in MSCBM-CD (Lonza) at a concentration of 2 μg/μL (LYO);
hPDLSCs treated for 24 h with ultrapure LPS from Porphyromonas gingivalis (tlrl-ppglps, InvivoGen, San Diego, CA, USA), 5 μg ml−1 (LPS);
hPDLSCs treated for 24 h with LPS and LYO (LPS + LYO).
All samples were observed at inverted light microscopy DMIL (Leica Microsystem, Milan, Italy) to evaluate the morphological cell features.
5
CMTM5 Impact on Wound Healing Dynamics
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A wound healing assay was performed to assess the effect of CMTM5 on cell lateral migration. CMTM5-overexpressing cells and vector control cells were placed in 6-well plates (500 l/well). A scratch was created using a 100 µl-sterile micropipette tip. Cells were then washed twice with PBS to remove cellular debris and cultured at 37°C in serum-free DMEM for 24 h. Cells were imaged using an inverted light microscope (×20 magnification, DM IL; Leica Microsystems GmbH) at 0 and 48 h, and the wound width was analyzed using ImageJ software 1.46 (National Institutes of Health).
6
Titanium Disk Culturing of hPDLSCs
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Human PDLSCs were seeded on titanium disks, CTRL and TEST, with MSCGM-CD (Lonza). The culture medium was changed twice a week. Cultures were maintained for 8 weeks, then were processed as described below for further examination. The inverted light microscopy DMIL (Leica microsystem, Milan, Italy) was used to carried out the microphotographs in order to evaluate the morphological arrangement of hPDLSCs and titanium disks.
7
BMSC Cell Viability Assay
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The labeled cells were vaccinated in a 25-cm2 culture flask (Corning-Costar), and incubated at 37°C with 5% CO2. A total of 15 μl re-suspension liquid from labeled BMSCs was added to 4% trypan blue solution (Beyotime Institute of Biotechnology) at a 1:1 ratio. The inverted microscope (DM IL; Leica Microsystems) was used to count the total cells. As the blue-stained cells were the dead cells, the cell survival ratio was calculated as [1−(number of blue-stained cells/total number of cells)] ×100%. Every other day, survival tests were performed on cells from the experimental and the control group. Three samples per group were examined, and counting of each sample was repeated four times. The cell numbers were counted, respectively between days 0–14 in order to construct cell growth curves, and the cell survival ratio was calculated for 24, 48 and 72 h.
8
Evaluating Plastic Adherence of hPDLSCs
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To evaluate plastic adherent ability, hPDLSCs at P2 were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 hours, subsequently stained with toluidine blue and observed by light microscopy (DMIL; Leica Microsystems, Wetzlar, Germany).
9
Wound Healing Assay Using EC109 and EC9706 Cells
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EC109 and EC9706 cells were seeded into six‐well plates and scratched using a 1 ml tip after cells formed a confluent monolayer.33 The closure of the wound was analyzed under a microscope (DM IL, Leica Microsystems), and images were collected at 0 and 24 h. Image J software was utilized to quantify and compare the relative migration rate between groups.
10
Characterization of Human Periodontal Ligament Stem Cells
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Cells derived from periodontal tissue were collected as previously described, scraping a third coronal root surface using Gracey’s curette33 (link). All three patients, included in the study, were in good general health and exempt from oral and systemic diseases. After collection, hPDLSCs were cultured using MSCGM-CD medium (mesenchymal stem cell growth medium chemically defined) (Lonza, Basel, Switzerland) and were maintained in an incubator at 37 °C in a humidified atmosphere of 5% CO2 in air. To evaluate cells morphological features, plastic-adherent hPDLSCs were stained used toluidine blue solution and then observed at inverted light microscopy DMIL (Leica Microsystem, Milan, Italy)34 .
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