A549 cells (3 × 105) were grown in the apical compartment of Transwell® polycarbonate-permeable supports (pore size 0.4 μm, Corning Life Sciences) at 37 °C and 5% CO2. After cells had adhered to the membrane, the media in both compartments was aspirated and replaced with DMEM containing 1% (v/v) Ultroser G and LL-37 at varying concentrations (0–50 μg/ml) in the presence and absence of HRV (MOI 10). Cells were incubated for 6 h at 33 °C and 5% CO2. Cells were then fixed in 10% neutral buffered formalin (3.7% formaldehyde) for 10 min, then washed once in PBS. Cells were then permeabilised in ice cold 0.1% Triton X-100/0.1% sodium citrate for 3 min and washed twice with PBS. An in situ cell death detection kit (Roche Applied Science, UK) was used to label cells according to the manufacturer’s instructions. Transwell membranes were excised with a scalpel and mounted on a glass slide in 50 μl Vectashield Hardset (containing DAPI). At least four random fields of view were counted, each containing more than 80 cells. The total number of DAPI-positive nuclei was assessed for each treatment to evaluate total cell number and the number of TUNEL-positive cells is expressed as a percentage of the total number of DAPI-positive nuclei.
Transwell polycarbonate permeable supports
Manufactured by Corning
Sourced in United States
Transwell polycarbonate-permeable supports are a type of laboratory equipment used for cell culture studies. These supports consist of a polycarbonate membrane with pores, which allow for the passage of cells, nutrients, and other materials between the upper and lower chambers of the Transwell system.
Automatically generated - may contain errors
Lab products found in correlation
In situ cell death detection kit, by Roche (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Recombinant murine cxcl1, by Thermo Fisher Scientific (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Merck Group (1 mentions)
7 protocols using transwell polycarbonate permeable supports
1
LL-37 Effect on HRV-Induced Cell Death
2
Bovine Brain Endothelial Cell Culture
3
MDSC Migration Assay with CXCL1 and CXCR2 Antagonist
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In vitro migration of murine MDSCs was evaluated in 24-well plates with transwell polycarbonate-permeable supports (8.0 μm; Costar Corning, Cambridge, MA, USA). MDSCs (1 × 106/mL) were seeded in the upper chambers of the inserts 30 min after incubation with culture supernatants of the differential expression Hepa1-6 cells at each concentration. Next recombinant murine CXCL1 (PeproTech, Rocky Hill, NJ, USA) were placed in the lower chamber at a concentration of 0, 10, or 100 ng/mL. The number of MDSCs in the bottom compartment was counted 24 h later.
To evaluate the suppressed migration of MDSCs, MDSCs (1 × 106/mL) were plated in the upper chambers of the inserts 30 min after incubation with the CXCR2 antagonist at a concentration of 0, 100, or 1000 ng/mL at each concentration, the number of MDSCs in the bottom compartment was counted 24 h later. Next recombinant murine CXCL1 (PeproTech, Rocky Hill, NJ, USA) was placed in the lower chamber at a concentration of 10 ng/ml. After incubation for 24 h, the number of MDSCs in the bottom compartment was counted.
To evaluate the suppressed migration of MDSCs, MDSCs (1 × 106/mL) were plated in the upper chambers of the inserts 30 min after incubation with the CXCR2 antagonist at a concentration of 0, 100, or 1000 ng/mL at each concentration, the number of MDSCs in the bottom compartment was counted 24 h later. Next recombinant murine CXCL1 (PeproTech, Rocky Hill, NJ, USA) was placed in the lower chamber at a concentration of 10 ng/ml. After incubation for 24 h, the number of MDSCs in the bottom compartment was counted.
4
MDSC Migration Assay with Tumor Factors
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MDSC in vitro migration was evaluated in 24-well plates with transwell polycarbonate-permeable supports (8.0 μm) (Costar Corning, Cambridge, MA, USA). MDSCs (1.0 × 105; > 90% Gr1 + CD11b + ) were plated in 100 μL of MEMα in the upper compartment 30 min after incubation with anti-GM-CSFRα Ab (10 μg/ml, R&D, Minneapolis, MN, USA), or IgG control Ab, and 500 μL of chemoattractant (ID8-Vegf cell or HM-1 cell tumour-conditioned media (TCM)) was added to the lower compartment. Plates were incubated at 37 °C with 5% CO2 for 3 h, and the number of MDSCs in the bottom compartment was counted using CountBright™ Absolute Counting Beads (Life Technology, Carlsbad, CA, USA). To obtain TCM, supernatants were collected from confluent cultures of HM-1 or ID8-Vegf cells cultured in MEMα or RPMI 1640 containing 10% serum.
5
Transwell Assay for MDSC Migration
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In vitro migration of murine MDSCs was evaluated in 24-well plates with transwell polycarbonate-permeable supports (8.0 μm; Costar Corning, Cambridge, MA, USA). MDSCs (5 × 105; >80% purity) were plated in the upper chambers of the inserts 30 min after incubation with a CXCR2 antagonist at each concentration, and recombinant murine CXCL1, CXCL2, and CXCL5 (PeproTech) were placed in the lower chamber at a concentration of 1, 10, or 100 ng/mL. After incubation for 3 h, the number of MDSCs in the bottom compartment was counted using Acubright counting beads (Life Technologies, Carlsbad, CA, USA)17 (link).
To evaluate the migration of human MDSCs, MDSCs (1 × 105; >80% purity) were plated in the upper chambers of the inserts 30 min after incubation with the CXCR2 antagonist at each concentration, and recombinant human CXCL1, CXCL2, and CXCL5 (PeproTech) were placed in the lower chamber at a concentration of 1 or 10 ng/ml. After incubation for 6 h, the number of MDSCs in the bottom compartment was counted36 (link).
To evaluate the migration of human MDSCs, MDSCs (1 × 105; >80% purity) were plated in the upper chambers of the inserts 30 min after incubation with the CXCR2 antagonist at each concentration, and recombinant human CXCL1, CXCL2, and CXCL5 (PeproTech) were placed in the lower chamber at a concentration of 1 or 10 ng/ml. After incubation for 6 h, the number of MDSCs in the bottom compartment was counted36 (link).
6
In vitro Transwell Assay for Murine PMN-MDSC Migration
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In vitro migration of murine PMN-MDSCs was evaluated in 24-well plates with Transwell polycarbonate-permeable supports (8.0 μm; Costar Corning, Cambridge, MA). Freshly isolated splenic PMN-MDSCs (1 × 106) were seeded in the upper chambers of the inserts. The CM from Hepa 1-6, Hepa 1-6–OXA, Hepa 1-6–OXA–control, and Hepa 1-6–OXA–shMyc were placed in the lower chamber. The mouse CCL5 antibody was placed in the lower chamber at a concentration of 10 μg/mL (R&D Systems). After incubation for 36 hours, the number of PMN-MDSCs in the bottom compartment was counted.
7
Transwell Assay for Cell Migration and Invasion
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The ability of cell migration was evaluated using the Costar Transwell polycarbonate permeable supports. For the Transwell migration assay, a total of 10 5 cells resuspended in 100 µL of serum-free DMEM media were applied to the upper chamber of the device, and 600 µL of medium containing 10% FBS was added to the lower chamber. A polycarbonate membrane with a pore size of 8 µm was placed between the two chambers. Cells were allowed to incubate for 12-18 h at 37°C. The cells that had not yet migrated were removed from the upper surface of the membranes with cotton swabs. Adhered cells, attached to the lower surface of the membranes, were stained with 4′,6-diamidino-2-phenylindole (DAPI)-phosphate-buffered saline (PBS) to visualize the nuclei. Cell numbers in five predetermined fields in each replicate were counted under the microscope (OLYMPUS DP80). All assays were independently repeated at least for three times. Cell invasiveness was examined using a Matrigel basement membrane matrix invasion assay (Millipore) according to the manufacturer's instructions. Cell invasion assays were performed as the migration assays except the Transwell membrane was precoated with Matrigel (Millipore) and the cells were incubated for 18-24 h.
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