The cell proliferation activity was assessed by EdU (5-ethynyl-2′-deoxyuridine) incorporation and flow cytometry. The EdU assay was performed according to the manufacturer′s instructions (RiboBio, Guangzhou, China), and the percentage of EdU-positive cells was used to evaluate cell proliferation. For flow cytometry, the cells were stained with 2 µM CFSE (Sigma, USA). Data were analyzed using Cell Quest and ModFitLT software (BD, USA), and the proliferation index was calculated as previously described31 (link). For apoptosis, the cells were assayed using a FITC AnnexinV Apoptosis Detection Kit (BD, USA) followed by flow cytometry analysis. For in vitro transwell assays, 1 × 105 cells were seeded in the upper chamber of a culture insert (8-mm pore size in 24-well plates, BD, USA) without (migration assay) or with (invasion assay) Matrigel (BD, USA) in serum-free media. The bottom chamber was filled with the same medium supplemented with 10% fetal bovine serum. After 24 h, the cells on the undersurface of the membrane were stained with crystal violet and counted using a light microscope. The cell migration and invasion properties were evaluated by the number of cells per high power field. All experiments were performed in triplicate and with at least three biological replicates.
Culture insert
Manufactured by BD
Sourced in United States, France
The Culture Insert is a disposable, sterile laboratory product designed to facilitate cell culture and tissue engineering applications. It provides a three-dimensional matrix to support the growth and organization of cells in a controlled microenvironment.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (3 mentions)
Modfit lt, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Cellquest, by BD (1 mentions)
6 well plate, by BD (1 mentions)
Panc 1, by BD (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Panc 1, by RIKEN BioResource Center (1 mentions)
Culture insert, by Corning (1 mentions)
Adenine, by Corning (1 mentions)
Insulin, by Corning (1 mentions)
Rat tail collagen type 1, by Corning (1 mentions)
Triiodothyronine, by Corning (1 mentions)
7 protocols using culture insert
1
Cell Proliferation, Apoptosis, and Transwell Assays
2
Co-culture of Pancreatic Cancer Cells
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Pancreatic cancer cell lines (PANC-1) were purchased from RIKEN Bio-Resource Center (Tsukuba, Japan) and were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (0.1 mg/mL) at 37°C in a humidified atmosphere with 5% CO 2 . Human pancreatic stellate cells (HPaSteCs, PSCs) were bought from American Type Culture Collection (ATCC, MD, USA) and its culture medium was the same as PANC-1 cells.
Co-culture procedures were as follows: PANC-1 cells were seeded into a 6-well plate (BD Biosciences, NJ, USA) with a density of 1 × 10 5 cells/well and the PSCs were plated into a culture insert (BD Biosciences) with 1.0 μm pores at a density of 5 × 10 5 cells/insert. The culture insert plated with PSCs were placed into the 6-well plate seeded with PANC-1 cells at 37°C humidified incubator for 24 h. The PANC-1 cells co-cultured with PSC cells were regarded as PANC-Co cells.
Co-culture procedures were as follows: PANC-1 cells were seeded into a 6-well plate (BD Biosciences, NJ, USA) with a density of 1 × 10 5 cells/well and the PSCs were plated into a culture insert (BD Biosciences) with 1.0 μm pores at a density of 5 × 10 5 cells/insert. The culture insert plated with PSCs were placed into the 6-well plate seeded with PANC-1 cells at 37°C humidified incubator for 24 h. The PANC-1 cells co-cultured with PSC cells were regarded as PANC-Co cells.
3
Organotypic Skin Culture with PM2.5 Exposure
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To produce organotypic skin culture using HEKs, NIH 3T3 J2 murine embryonic fibroblasts (ATCC, CCL-92) and culture insert (BD Biosciences) were used. Murine fibroblasts were cultured in DMEM containing 4.5 g/L glucose (Corning). A mixture of fibroblasts and rat tail type I collagen (Corning) were plated onto the culture inserts as a dermal equivalent. HEKs (2 × 106/culture insert) were then plated on top of the dermal equivalent, air-lifted after 1 day, and cultured for 7 days in DMEM containing 1% of gentamicin/amphotericin and growth factors such as adenine, insulin, apo-transferrin, and triiodothyronine (Corning). The air-liquid interface organotypic skin cultures were stimulated with a vehicle control (culture medium with 0.1% DMSO) or PM2.5 (1 ng/mL) applied to the surface of the air-liquid interface culture for an additional 7 days, and then the skin was fixed with 4% buffered formalin for immunostaining.
4
Wound Healing Assay with Virus Infection
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A scratch wound was created using a Culture-Insert (BD Labware Europe, Le Pont De Claix, France). The BV2 cell monolayer (1.2 × 105 cells/well) was seeded into a 24-well cell culture plate and cultured overnight. Then, the insert was removed, washed with PBS, and placed into fresh medium prior to virus infection. The wound area of the BV2 monolayer was photographed 0 or 12 h post-infection. The number of cells that migrated into the cleared wound area were counted using ImageJ (http://imagej.nih.gov/ij/download.html ) and the reduction of the wound area was measured. The relative cell migration distance was analyzed using the Gradientech Tracking Tool (http://gradientech.se/tracking-tool-pro/ ). The results were automated to track the cell migration distance and velocity.
5
Transwell Migration Assay Protocol
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For transwell migration assay, culture insert (8 um pore size; BD bioscience, Inc., San Jose, California, USA) were precoated with matrigel (BD bioscience, Inc., San Jose, California, USA). 1x105 cells (in DMEM medium with 1% (v/v) fetal bovine serum) were applied to the insert. The bottom wells contained 600 uL of culture medium with 10% (v/v) fetal bovine serum and were left un-agitated in the cell incubator. After 24 hrs, the cells attached to the transwell membrane were fixed with 0.5% (w/v) crystal violet and washed extensively. The stained cells were visualized and counted under microscopy.
6
Scratch Wound Migration Assay
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A scratch wound was created using a Culture-Insert (BD Labware Europe, Le Pont De Claix, France) following the procedures described in our previous study [17 (link)]. The wound was photographed at 0 and 12 h post-infection, and the cells that migrated into the cleared wound area were counted using ImageJ (http://imagej.nih.gov/ij/download.html ) and the reduction of the wound area was measured.
7
Transwell Migration Assay Protocol
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For transwell migration assay, culture insert (8 um pore size; BD Company, Franklin Lakes, NJ, USA) were precoated with matrigel (BD Company, Franklin Lakes, NJ, USA). 1×105 cells (in DMEM medium with 1% (v/v) FBS) were applied into the insert. The bottom wells contained 600 uL of culture medium with 10% (v/v) FBS, and were left un-agitated in the cell incubator until observation.
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