Cell migration was performed using 8 µm pore polycarbonate membranes (Costar, Cambridge, MA). Ad‐MSCs were loaded at 50,000 cells per insert (upper chamber) and PRP gel was added to the lower chamber in DMEM F12 (1:1) 0.25% BSA. The cells were allowed to migrate into the lower chamber at 37 C in a 5% CO2 atmosphere saturated with H2O for 24 h in presence of mitomycin C (10 mg/ml; Sigma–Aldrich). At the end of incubation, cells that had migrated to the lower side of the filter were fixed with 11% glutaraldehyde for 15 min at room temperature, washed three times with PBS, and stained with 0.1% crystal violet‐20% methanol for 20 min at room temperature. After three PBS washes and complete drying at room temperature, the crystal violet was solubilized by immersing the filters in 10% acetic acid. The concentration of the solubilized crystal violet was evaluated as absorbance at 540 nm.
8 m pore polycarbonate membrane
Manufactured by Corning
Sourced in United States
The 8µm pore polycarbonate membranes are a type of lab equipment used for filtration and separation purposes. They are made of polycarbonate material and have a pore size of 8 micrometers. The core function of these membranes is to facilitate the separation and purification of materials based on their size.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Leica (2 mentions)
Mitomycin c, by Merck Group (1 mentions)
Transwell insert, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Apc annexin 5 apoptosis detection kit with pi, by BioLegend (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
8 protocols using 8 m pore polycarbonate membrane
1
Quantifying Cell Migration using Crystal Violet
2
Transwell-based Cell Migration and Invasion
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Cell migration and invasion were assessed in transwells with 8µm-pore polycarbonate membranes (Costar) as previously described (20) .
Cell invasion was measured in the same way, except that prior to cell seeding the upper chamber was prepared by coating the filter with 100l Matrigel at 0.3mg/mL (BD Biosciences) and the cells attached to the lower surface were fixed, stained and counted. Further details including wound healing assay is provided in the supplementary data.
Cell invasion was measured in the same way, except that prior to cell seeding the upper chamber was prepared by coating the filter with 100l Matrigel at 0.3mg/mL (BD Biosciences) and the cells attached to the lower surface were fixed, stained and counted. Further details including wound healing assay is provided in the supplementary data.
3
Evaluating Breast Cancer Metastasis
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Migration and invasion assays were performed to evaluate the effect of MIR4435-2HG on breast cancer cell metastasis. Briefly, the Transwell inserts (Costar; Corning, Inc.) with 8 µm pore polycarbonate membranes (Corning, Inc.) coated with Matrigel at 37°C for 4 h or without Matrigel (BD Biosciences) were used in the invasion and migration assays, respectively. MDA-MB-231 and MCF-7 cells infected with shNC or shMIR4435-2HG viruses were resuspended in a serum-free medium and further 6×104 cells for MCF-7 or 2×104 cells for MDA-MB-231 were seeded into the upper chamber of the Transwell insert. The lower chamber was filled with 500 µl DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). After 24 h incubation at 37°C with 5% CO2, cells in the upper chamber were gently removed using a cotton swab, whereas cells in the lower chamber were washed with PBS, fixed with 4% paraformaldehyde at 4°C for 20 min, and stained with 0.1% crystal violet at room temperature for 20 min. Eventually, cells that had migrated/invaded into the filter were counted and analyzed. Images of the representative migrative or invasive pictures were captured using a light microscope (magnification, ×100). Each assay was performed in triplicate.
4
Macrophage Chemotactic Migration Assay
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Macrophage chemotactic migration assay was performed using a 24-well Transwell system, with upper and lower culture chambers separated by 8µm pore polycarbonate membranes (Corning, USA), as previously described [18] . The bottom chamber was lled with IL-34 protein in Dulbecco's modi ed eagle's medium (DMEM) (Hyclone, USA) containing 10% fetal bovine serum (Gibco, USA) or CM from TNBC cells.
In the upper chambers, macrophages (RAW264.7, 5 × 10 4 cells/well) suspended in DMEM were seeded and then incubated for 24 h. Macrophages that migrated to the lower surface of the membrane were xed in 4% paraformaldehyde, stained with Giemsa, and then photographed and counted with a light microscope (Leica). To assess the IL-34-induced p38 and mTOR signaling pathways, the cells were treated with selective inhibitors. The chemotactic index was calculated as the number of macrophages that migrated to IL-34 of the CM from TNBC cells divided by the number of macrophages that migrated to DMEM alone [19] .
In the upper chambers, macrophages (RAW264.7, 5 × 10 4 cells/well) suspended in DMEM were seeded and then incubated for 24 h. Macrophages that migrated to the lower surface of the membrane were xed in 4% paraformaldehyde, stained with Giemsa, and then photographed and counted with a light microscope (Leica). To assess the IL-34-induced p38 and mTOR signaling pathways, the cells were treated with selective inhibitors. The chemotactic index was calculated as the number of macrophages that migrated to IL-34 of the CM from TNBC cells divided by the number of macrophages that migrated to DMEM alone [19] .
5
Macrophage Chemotactic Migration Assay
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Macrophage chemotactic migration assay was performed using a 24-well Transwell system, with upper and lower culture chambers separated by 8µm pore polycarbonate membranes (Corning, USA), as previously described [18] . The bottom chamber was lled with IL-34 protein in Dulbecco's modi ed eagle's medium (DMEM) (Hyclone, USA) containing 10% fetal bovine serum (Gibco, USA) or CM from TNBC cells.
In the upper chambers, macrophages (RAW264.7, 5 × 10 4 cells/well) suspended in DMEM were seeded and then incubated for 24 h. Macrophages that migrated to the lower surface of the membrane were xed in 4% paraformaldehyde, stained with Giemsa, and then photographed and counted with a light microscope (Leica). To assess the IL-34-induced p38 and mTOR signaling pathways, the cells were treated with selective inhibitors. The chemotactic index was calculated as the number of macrophages that migrated to IL-34 of the CM from TNBC cells divided by the number of macrophages that migrated to DMEM alone [19] .
In the upper chambers, macrophages (RAW264.7, 5 × 10 4 cells/well) suspended in DMEM were seeded and then incubated for 24 h. Macrophages that migrated to the lower surface of the membrane were xed in 4% paraformaldehyde, stained with Giemsa, and then photographed and counted with a light microscope (Leica). To assess the IL-34-induced p38 and mTOR signaling pathways, the cells were treated with selective inhibitors. The chemotactic index was calculated as the number of macrophages that migrated to IL-34 of the CM from TNBC cells divided by the number of macrophages that migrated to DMEM alone [19] .
6
Evaluating hMSC-derived Factors on GBM Survival
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To evaluate the effect of soluble factors derived from hMSCs on the survival of GBM cells, U251 cells were co-cultured with CH-hMSCs and PL-hMSCs using a transwell. U251 cells were seeded in a 24-well plate at a density of 3 × 104 cells/well in 500 µl DMEM + 10% (v/v) FBS and cultured for 12 h. At this stage, 3 × 104 hMSCs resuspended in 250 µl DMEM + 10% FBS were seeded into a transwell insert with an 8 µm pore polycarbonate membrane (Corning, U.S.A.). The transwell inserts were then placed in the 24-well plates containing U251 cells, the volume of growth medium in the lower chamber was adjusted to 750 µl. After 72 h of culture, transwell inserts were discarded, U251 cells were harvested, and percentages of apoptotic U251 cells were determined using the APC Annexin V Apoptosis Detection Kit with PI (Biolegend, U.S.A.) according to the manufacturer’s instructions. Signal detection was performed by FACS Calibur™ (Becton Dickinson, U.S.A.) using Cell Quest® software (Becton Dickinson, U.S.A.).
7
Evaluating Fordin's Effect on Cell Invasion
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The effect of fordin on cell invasion ability was investigated using a Transwell assay. Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) diluted 1:8 in cold serum-free culture media was added to each 24-well Transwell chamber with an 8-µm pore polycarbonate membrane (Corning Incorporated, Corning, NY, USA) and incubated under standard conditions for at least 12 h to gel. The cells treated with fordin (1 µM and IC20) for 24 and 48 h were seeded onto the Matrigel. Each lower chamber contained 600 µl of culture media containing 10% FBS. Following incubation for 24 h, transmembrane cells were stained by 0.1% crystal violet. Six fields were randomly selected under the inverted fluorescence microscope, and the number of transmembrane cells was counted.
8
Cell Migration Assay in HUVECs
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Transwell assay was employed to determine cell migration in HUVECs [12, (link)11] (link). Brie y, after being transfected with miRNA or siRNA particles, cells in 200 µL 1% FBS medium were placed for 30 min in a modi ed Boyden chamber with an 8 µm-pore polycarbonate membrane (Corning, USA) pre-coated with serum-free medium. The lower chamber contained 600 µL 1% FBS medium. Plates were incubated at 37℃ in 5% CO 2 for 12 h. Then the chambers were washed with PBS three times and xed with 4% paraformaldehyde (Sigma-Aldrich, USA). Crystal violet (Sigma-Aldrich, USA) was used to stain cells for 15 min. After rinsing thoroughly, non-migrated cells on the upper membranes were wiped off with cotton swabs. The migrated cells of the membrane were photographed by microscopy and counted with Image J software (NIH, USA).
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