Senescent EPCs (2 × 105 cells/well, passage 20) were pretreated with DMSO, 10 nM MHY2233, 100 nM resveratrol, or 100 nM EX527 and seeded in 6-well plates. Cell migration was assessed by the scratch wound healing method. After 24 h of culturing, at full confluence, a linear gap in the cell monolayer was created by scratching the surface with an SPLScar Scratcher (SPL Life Science, Korea). The cells were washed to remove detached cells and incubated at 37°C. Images were captured by using a light microscope (OLYMPUS, Tokyo, Japan) at 0, 2, 4, and 6 h. The migrated area was calculated using the following formula:
Splscar scratcher
Manufactured by SPL Life Sciences
Sourced in Cameroon
The SPLScar Scratcher is a laboratory instrument designed for the gentle and controlled removal of cell monolayers. It features a motorized, adjustable scratching mechanism that can create uniform scratch wounds in cell culture dishes. The device is intended for use in in vitro wound healing and cell migration studies.
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Lab products found in correlation
Gene5, by Agilent Technologies (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Inverted microscope, by Zeiss (2 mentions)
Cytation 3 cell imaging multi mode reader, by Agilent Technologies (2 mentions)
Dmi8 inverted light microscope, by Leica (1 mentions)
Cell imaging multi mode reader cytation, by Agilent Technologies (1 mentions)
Dmil led inverted microscope, by Leica (1 mentions)
6 well plate, by SPL Life Sciences (1 mentions)
Ckx41 microscope, by Olympus (1 mentions)
Eclipse ts2, by Nikon (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Cytation 3 cell imaging reader, by Agilent Technologies (1 mentions)
Rhil 33, by R&D Systems (1 mentions)
Ckx31, by Olympus (1 mentions)
Cell stain solution, by Merck Group (1 mentions)
Chemicon cell invasion assay kit, by Merck Group (1 mentions)
Envision 2105, by PerkinElmer (1 mentions)
Matrigel, by Corning (1 mentions)
Inverted microscope, by Olympus (1 mentions)
30 protocols using splscar scratcher
1
Senescent EPC Migration Assay
2
Scratch Assay for Cell Migration
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For the scratch migration assays, the cells were seeded in 6-well plates and grown until confluence in complete EGM-2 medium. A linear gap was created by scratching the cells using a SPLScar Scratcher (SPL Life Science, Korea). The cells were washed to remove any detached cells and incubated at 37°C for 6 h. Cell migration activity was expressed as the relative healing area according to the following formula: [(original scratch area - new scratch area)/original scratch area] × 100%.
3
Wound Healing Assay with Selective Estrogen Receptor Modulators
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Nthy/WT and Nthy/V600E were seeded at 5E4 cells per each well in 24-well culture plate and incubated until all wells reach 100% confluence. Each well was wounded by scratching with a SPLScar scratcher (#201925, SPL Life Science, Pocheon, Korea) and washed with Dulbecco’s phosphate-buffered saline (DPBS) with Ca2+ and Mg2+ to remove the debris followed by filling up 400 μL of complete media with 10% FBS and phenol red. After confirming that wounds are evenly formed without a chunk of cells, we added 100 μL of media containing either DMSO, 50 nM of diarylpropionitrile (DPN; #1494, ERβ-selective agonist, Tocris Bioscience, Bristol, UK), or 5 μM of methylpiperidino pyrazole (MPP; #13863, ERα-selective antagonist, Cayman Chemical, Ann Arbor, MI, USA). The concentrations were determined based on the previous studies using these molecules [30 (link)-32 (link)]. We marked the clearly scratched area under the plates before incubation and took pictures after 0 and 6 hours of incubation. In the two matched images, we made two representative borders for before and after incubation with black and red colors, respectively. The difference of widths between the borders was measured for each condition to compare the relative migrated area.
4
Automated Wound Healing Assay
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For the wound-healing assay, cells were cultured in 24-well plates (1 × 105 cells per well) for 24 h to achieve 90% confluence. A vertical or horizontal wound was created by using a SPLScar scratcher (SPL, Korea). The wounded cells were washed once with DMEM or RPMI 1640 with 10% FBS and allowed to grow in the medium for 24–48 h. Images of cells were taken with a Leica DMi8 inverted light microscope and quantitatively analyzed using ImageJ 1.51s software (https://imagej.nih.gov/ ) installed with a macro (edge-detection/thresholding method) for automated analysis of scratch-wound assays.
5
Evaluating SCC15 Cell Migration
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Cell migration of the SCC15 cell line was evaluated by wound closure assay modified from a previous method [32 (link)]. The SCC15 cells were seeded into 6-well plates at a density of 1x106 cells/ml and incubated at 37°C until reaching 80% confluence as a monolayer. The cell monolayer was scraped in a straight line with a SPL scar scratcher (SPL Life Sciences, Korea). Detached cells were removed and washed twice with 1 ml of medium. Cells were treated as described in the colony formation assay and incubated for 36 hours. A snapshot of cells from each condition was taken at time points of 6, 12, 24 and 36 hours using an inverted microscope (Carl Zeiss Microscopy GmbH, Germany). Distance of the wound area was analyzed by the ImageJ system.
6
Scratch Wound Cell Migration Assay
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Cultured cells grown into confluent monolayers in 12-well plates were scraped with a sterilized SPLScar Scratcher (SPL Life Sciences, Pocheon, Korea), and then washed extensively with 1× PBS to remove nonadherent cells. The remaining attached cells were continuously cultured in DMEM supplemented with 1% FBS at 37ºC, and monitored for cell migration from 24 to 48 hours.
7
Wound Healing Assay for PRDX2 Overexpression and Knockdown
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PRDX2-overexpressing ORL-48T cells were seeded into 24-well tissue-culture plates at a density of 150,000 cells per well. After incubation for 24 hours, a linear gap was created by scratching the bottom of each well using a SPLScar Scratcher (SPL Life Science, Korea). The cells were then maintained in DMEM/F12 medium supplemented with 1% FBS and antibiotics for 24 and 48 hours. The extent of wound closure was determined using NIS-Elements Advanced Research Imaging Software version 3.0. This was converted into a percentage that was relative to the initial width of the wound. Three independent replicates of each experiment were performed.
For PRDX2-knockdown, ORL-48T cells were seeded into 24-well plates at 150,000 cells per well. Thereafter, cells were transfected using Lipofectamine 2000 with either 100 nM siRNA-Control or 100 nM siRNA-PRDX2 according to manufacturer’s instructions. After transfection, cells were incubated overnight, then cells were maintained in DMEM/F12 medium supplemented with 1% FBS and antibiotics and prepared as above.
For PRDX2-knockdown, ORL-48T cells were seeded into 24-well plates at 150,000 cells per well. Thereafter, cells were transfected using Lipofectamine 2000 with either 100 nM siRNA-Control or 100 nM siRNA-PRDX2 according to manufacturer’s instructions. After transfection, cells were incubated overnight, then cells were maintained in DMEM/F12 medium supplemented with 1% FBS and antibiotics and prepared as above.
8
Wound Closure Assay for HaCaT Cells
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HaCaT cells, at a seeding density of 1 × 105 cells/well (500 μL), were cultured in 24-well plates in complete DMEM and allowed to proliferate until they reached 95–100% confluency. Cells were then serum-starved for 24 h in DMEM without the presence of FBS. A SPLScar™ Scratcher (SPL Life Sciences, Gyeonggi-do, Republic of Korea) was used to scrape the cell monolayer (in a vertical and horizontal scratch lines) to create an in vitro cell monolayer wound model in each well. The cells were washed once with sterile PBS to clear any debris and floating cells, and then the well was refilled with serum-free DMEM or DMEM containing DMSO (vehicle control), PC at 62.5 μM, EGF at 1 ng/mL, or the combination of PC and EGF. HaCaT monolayer wound closure was monitored under a phase-contrast microscope at 0 h, 24 h, 48 h and 72 h. Percent closure of the scratch at each time point for different treatment conditions was then quantified. Each treatment was performed in triplicate, and each experiment was repeated at least three times.
9
Cell Scratch Assay for Migration
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Cell migration was analyzed by cell scratch assay [38 (link)]. Briefly, hADSCs were seeded into 24-well tissue culture plates at a density of 1.5 × 104 cells/well. SPLScar scratcher (SPL Life Sciences, Pocheon, Korea) was used to scratch cells prior to green OLED irradiation. Cell-covered area was estimated as follows: [1 − (pixel size of uncovered area at each time point)/(pixel size of initially scratched area)] × 100%[39 (link)].
10
Evaluating Cell Migration Using Scratch Assay
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Scratch assay was used for evaluating of migratory properties of tested cells under specific conditions. HeLa cells (3.5 × 105) were seeded into 6-well plates and cultured in a growth medium until they reach confluence. A linear scratch through cell monolayer was made in individual wells using an SPL Scar™ scratcher (SPL Life Science, Pocheon, Korea). Each well was then gently washed 2 times with 3 mL of PBS and treated with L1 (1 µM and 10 µM) in the growth medium. Before the recording of the wounded area at 0 h, 24 h, 48 h, and 72 h, cells were stained with Hoechst 33342 (Sigma Aldrich, St. Louis, MO, USA) and captured with Cytation™ Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). The changes of wounded areas were determined also by cell count analysis using Gene 5 software (Biotek, Winooski, VT, USA).
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