The cells were grown in silicone Culture-Inserts 4 Well (80469, Ibidi, Gräfelfing, Germany) to create the cell-free area (wound). After reaching confluence, the inserts were removed and cells washed with PBS before preincubation with the inhibitors for 30 min and stimulated with LPA or EtOH. Pictures were taken at a 10x magnification from four different fixed areas with a Leica DMI3000B microscope (Leica Microsystems, Wetzlar, Germany) using Leica Application Suite (LAS) V4.7 at time points 0 and 8 h. The cell-free area was analyzed and calculated using ImageJ (NIH) software.
4 well culture inserts
Manufactured by Ibidi
Sourced in Germany
The Ibidi 4-well culture inserts are designed for cell culture applications. They provide a multi-well platform for growing and maintaining cells in a controlled environment. The inserts are made of tissue culture-treated polystyrene and feature four individual wells, allowing for parallel experiments or replicates within a single device.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmi3000b microscope, by Leica (1 mentions)
Incucyte zoom, by Sartorius (1 mentions)
Slide angiogenesis, by Ibidi (1 mentions)
Axitinib, by MedChemExpress (1 mentions)
Hislink protein purification resin, by Promega (1 mentions)
Irdye 800cw streptavidin, by LI COR (1 mentions)
Detoxi gel, by Thermo Fisher Scientific (1 mentions)
Proteome profiler human angiogenesis antibody array, by R&D Systems (1 mentions)
Hispur cobalt resin, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Mycoalert plus mycoplasma detection kit, by Lonza (1 mentions)
Celltiter 96 aqueous one solution, by Promega (1 mentions)
24 well cell culture insert, by BD (1 mentions)
Matrigel, by BD (1 mentions)
Mouse anti human gapdh antibody, by Abcam (1 mentions)
Goat anti mouse igg h l, by Abcam (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
6 protocols using 4 well culture inserts
1
Wound Healing Assay with Inhibitors
2
Wound Healing Assay for Transfected Cells
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A total of 4 × 104 of stably transfected cells were seeded into a 12-well plate with Culture-Inserts 4 Well (Ibidi, Gräfelfing, Germany). After culturing overnight, the well was removed, and the cells were incubated with a serum-free medium for another 24–48 h. Images of the wound healing process were captured at every 2 h using Incucyte ZOOM (Essen BioScience, Michigan, MI, USA). To evaluate the cells’ wound healing ability, the percentage of wound closure was calculated using ImageJ (National Institutes of Health, Bethesda, MD, USA).
3
HUVEC Culture and Angiogenesis Assays
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Medium 200 (M200PRF500), low serum growth supplement (LSGS; S00310), 0.025% trypsin-EDTA (R001100), VEGF (PHC9344), Pierce Protease Inhibitor Mini Tablets (A32953), HisPur™ Cobalt Resin (89964), Detoxi-Gel™ (20339), penicillin-streptomycin (15140122), amphotericin B (15290018), Qubit™ Protein Broad Range kit (A50668) and human umbilical vein endothelial cells (HUVECs; C0035C; RRID: CVCL K312 ) were purchased from Thermo Fisher Scientific/Fisher Scientific. ToxinSensor™ LAL Endotoxin Assay Kit (L00350) was purchased from Genscript. HisLink™ Protein Purification Resin (V8823) and CellTiter 96® AQueous One Solution (G3582) were purchased from Promega. Mitomycin C (M4287) was purchased from Sigma Aldrich. Proteome Profiler™ Human Angiogenesis Antibody Array (ARY007) was purchased from R&D Systems. IRDye 800 CW Streptavidin (926-32230) was purchased from LI-COR. Growth factor reduced Matrigel (GFR-Matrigel; 356231) was purchased from Corning. Axitinib (HY-10065) was purchased from MedChemExpress. 0.1 mm glass beads (P000929LYSK0A.0) and soft tissue homogenizing kits (P000933-LYSK0-A) were purchased from Bertin Corp. 4-well culture inserts (80466) and angiogenesis slides (81506) were purchased from ibidi. MycoAlert™ Plus Mycoplasma Detection Kit (LT07-701) was purchased from Lonza.
4
Transwell and Wound Healing Assays
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Transwell assays were performed with 24‐well cell culture inserts with an 8.0 µm pore size (Falcon, USA) with Matrigel (1 : 10) (BD Biosciences, USA). A total of 5 × 104 DLD1 or 8 × 104 HCT116 cells were placed into the upper chamber in 0.2 ml of serum‐free DMEM, whereas CAF cells or CAF CM or control medium was placed in the lower chamber as a chemoattractant. Migrated and invaded cells on the other side of the membrane were fixed and stained with crystal violet for 5 min after 24 h of culture. Each assay was repeated three times. For the wound healing assay, 5 × 104 cells per well in 4‐well culture inserts (ibidi, Germany) were seeded into 12‐well plates, and wounds were made. The size of the wound was captured every day by an IncuCyte ZOOM live cell imager and measured by ImageJ software.
5
Antibody Analysis in Cell Assays
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Rabbit anti-human LAMA1 antibody (sc-5582) was from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse anti-human GAPDH antibody, goat anti-rabbit IgG H&L (ab150077), and goat anti-mouse IgG H&L were purchased from Abcam (Cambridge, MA, USA). TRIzol reagent and DAPI (4′,6-diamidino-2-phenylindole) were obtained from Invitrogen (Carlsbad, CA, USA). The PrimeScript RT reagent kit with gDNA Eraser and SYBR Premix Ex Taq™ II (Tli RNaseH Plus) kit were from Takara Clontech (Kyoto, Japan). Cell Counting Kit-8 (CCK-8) was from Dojindo (Kumamoto, Japan), and 4-well culture inserts were from Ibidi (cat. number 80469; Martinsried, Germany).
6
Osteoblast-Osteoclast Co-culture Protocol
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Osteoblasts isolated from the trabecular bone of healthy individuals with the informed consent of the donors were obtained from PromoCell (#C12720), operating under Approval # 219-04 of the Ethics Commission of the State Chamber of Medicine. The cells were cultured according to the manufacturer’s instructions. Osteoclast precursors were derived from primary human monocytes by 6 days of culture in M-CSF as described above. Osteoblasts and osteoclast precursors were cultured in 35 mm dishes with 4-well culture inserts (ibidi, #81156) at a 3:1 well ratio. 48 h before co-culture mixing, osteoblasts were switched to serum free alpha MEM with 1x pen/strep (Gibco). Following serum starvation, 4-well culture inserts were removed, and cells were cultured in their conditioned media overnight with or without treatment. Cells were fixed with 4% paraformaldehyde the following morning.
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