RMF were plated in 0.4μm pore Transwell dishes (Costar) and co-cultured with vHMEC expressing either control luciferase short hairpin (sh-Luciferase), activin A short hairpin (sh-activin A), vector control, TRF2 or hTERT (19 (link), 20 (link)). RNA was isolated from RMF 48 hours after initiation of co-culture.
Transwell dishes
Manufactured by Corning
Sourced in United States
Transwell dishes are a type of cell culture insert consisting of a porous membrane that separates the top and bottom chambers. This configuration allows for the study of cell-cell interactions and the passage of substances across the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (2 mentions)
Anti cyr61 sc 13100, by Santa Cruz Biotechnology (2 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions)
Human umbilical vein endothelial cells (huvec), by Thermo Fisher Scientific (1 mentions)
Ckx41, by Olympus (1 mentions)
Mowiol, by Merck Group (1 mentions)
Cellsens standard software, by Olympus (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Vybrant did cell labeling dye, by Thermo Fisher Scientific (1 mentions)
Minimacs system, by Miltenyi Biotec (1 mentions)
Anti tubulin g436, by Bioworld Technology (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ammonium chloride potassium ack lysing buffer, by Lonza (1 mentions)
Recombinant human interleukin il 6, by R&D Systems (1 mentions)
Erlotinib, by Selleck Chemicals (1 mentions)
Anti mil 6, by R&D Systems (1 mentions)
17 protocols using transwell dishes
1
Co-culture of RMF and vHMEC for RNA Isolation
2
Transwell Assay for Cell Transmigration
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Transmigration was measured in transwell dishes (Corning-Costar, Lowell MA, 8 µm pore size, 6.5 mm diameter). Inserts were either used uncoated or coated with 0.1% gelatine (Sigma) for 30 min and 3.5 × 104 HUVEC (Cellworks, ZHC2301) for 3 days. For transmigration through HUVEC, cells were stained with 1 µM (SkMel2) or 10 µM (B16) Cell Tracker Green CMFDA (Life Technologies) for 30 min and recovered in DMEM with 10% FCS for 3 h (SkMel2) or 12 h (B16). Cells were detached using Versene and incubated in DMEM or HUVEC medium for 30 min. In all, 5 × 104 SkMel2 or B16 or 2 × 105 U87 cells were seeded on top of HUVEC, 106 (SkMel) or 5 × 105 (SNU-1) on the transwell insert, incubated as indicated and fixed with 4% formaldehyde. Cells on the top of the membrane were removed, transmigrated cells were stained with DAPI and membranes were mounted on a glass slide using Mowiol (Merck). Seven random images were obtained of DAPI (for transmigration through membrane only) or Cell Tracker Green (for transmigration through HUVEC) using a 10× objective on an Olympus CKX41 with an Olympus XM10 camera using the CellSens Standard software (Olympus). Cell numbers were quantified using ImageJ46 . All n values represent biological replicates performed as technical duplicates and averaged; all experiments were normalised to an untransfected control.
3
HUVEC-PBMC Transmigration Assay
4
Tracking Metastatic Potential of DAB2IP-Knockdown PC3 Cells
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Early study has shown that DAB2IP gene knockdown in PC3 cell, a poorly-metastatic line, increases its metastatic potential (also called as DAB2IP-knockdown PC3 cells or KD cells)24 . These stable cell lines expressing dual reporter genes (GFP and luciferase) were maintained in RPMI1640 medium (Invitrogen, Carlsbad, CA, USA) containing 5% PBS as previous described24 . Migration assays were performed in Transwell dishes (Corning Costar, Cambridge, MA, USA) as described earlier73 (link). For tracking the cell migration in vivo, both PC3 and KD were labeled with Qtracker® labeling kit (Life technologies, Carlsbad, CA, USA) by following manufacture's instruction as illustrated earlier74 (link). For histological study, Vybrant DiD cell labeling dye (Thermo Fisher Scientific, Waltham, MA, USA) was used to track cancer cells according to the product information for observation under microscope75 (link).
5
Differentiation of Caco2 Cells
6
Neutrophil Migration Assay for Leishmania
7
Investigating CYR61 in Gastric Cancer
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IHC staining S-P kit (KIT-9710) was purchased from MAIXIN Biology Corporation. Atorvastatin calcium was purchased from WuXi Sigma. Transwell dishes were purchased from Corning Inc. Matrigel was purchased from BD Biosciences. The gastric cancer cell line AGS was purchased from ATCC. Anti-CYR61 (SC-13100) was purchased from Santa Cruz, anti-Tubulin (G436) from Bioworld. The CYR61 shRNA oligos were synthesized by ShengGong Company.
8
Isolation and Culture of Airway Progenitors
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Airway epithelial progenitors were isolated from adult tracheas of wild type mice
or E2f4 mouse mutants and cultured using well-established protocol (see
Methods)41 (link)42 (link). For the functional analysis of E2f4 in ALI
cultures we used adult E2f4+/+,
E2f4+/f;
R26CreERT2/LSLZsGreen1or E2f4f/f;
R26CreERT2/LSLZsGreen1 mice, as well as mice
lacking the R26LSLZsGreen1 allele. Briefly, after treatment with
0.5% pronase overnight, cells were cultured on collagen1-coated Transwell
dishes (Corning) under submerged conditions in media that allowed expansion of
airway progenitors42 (link) until confluence (7 days). The ALI was
established by removing media from the upper chamber of Transwell (Day 0 ALI)
and culturing cells in differentiation media (mTEC/serum free, RA media)42 (link) up to 8 days (Day 8 ALI). Treatment with 1 μM
4-hydroxytamoxifen (Tm) from day −5 to day 0 was used to induce Cre
mediated recombination36 (link). The efficiency of recombination was
analysed by qPCR, IF for E2f4 and expression of ZsGreen1, where appropriate.
or E2f4 mouse mutants and cultured using well-established protocol (see
Methods)41 (link)42 (link). For the functional analysis of E2f4 in ALI
cultures we used adult E2f4+/+,
E2f4+/f;
R26CreERT2/LSLZsGreen1or E2f4f/f;
R26CreERT2/LSLZsGreen1 mice, as well as mice
lacking the R26LSLZsGreen1 allele. Briefly, after treatment with
0.5% pronase overnight, cells were cultured on collagen1-coated Transwell
dishes (Corning) under submerged conditions in media that allowed expansion of
airway progenitors42 (link) until confluence (7 days). The ALI was
established by removing media from the upper chamber of Transwell (Day 0 ALI)
and culturing cells in differentiation media (mTEC/serum free, RA media)42 (link) up to 8 days (Day 8 ALI). Treatment with 1 μM
4-hydroxytamoxifen (Tm) from day −5 to day 0 was used to induce Cre
mediated recombination36 (link). The efficiency of recombination was
analysed by qPCR, IF for E2f4 and expression of ZsGreen1, where appropriate.
9
Porcine Epithelial Cell Barrier Integrity Assay
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Intestinal porcine epithelial cells (IPEC-1) were a gift from Dr. Guoyao Wu (Texas A&M University). The cells were cultured in DMEM supplemented with 10% inactivated fetal bovine serum (Gibco) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin sulfate) at 37°C under 5% CO2 in a humidified incubator.
IPEC-1 cells were cultured in Transwell dishes (Corning Life Sciences, Corning, NY) for 21 d as before [24 (link)]. Monolayers of IPEC-1 cells were incubated for 6 h in DMEM without serum or antibiotics, in the presence of ETEC K88 (1 × 107 CFU), L. reuteri LR1 (1 × 108 CFU), or both, in the upper chamber. Permeability of the IPEC-1 cell monolayers was measured with FITC-dextran (4400 Da, Sigma-Aldrich, St Louis, MO) [24 (link)]. IPEC-1 cells were collected for enumeration of ETEC K88, real-time PCR (qPCR), and Western blotting analysis. Six wells per treatment were used, and the results were representative of 3 independent experiments.
IPEC-1 cells were cultured in Transwell dishes (Corning Life Sciences, Corning, NY) for 21 d as before [24 (link)]. Monolayers of IPEC-1 cells were incubated for 6 h in DMEM without serum or antibiotics, in the presence of ETEC K88 (1 × 107 CFU), L. reuteri LR1 (1 × 108 CFU), or both, in the upper chamber. Permeability of the IPEC-1 cell monolayers was measured with FITC-dextran (4400 Da, Sigma-Aldrich, St Louis, MO) [24 (link)]. IPEC-1 cells were collected for enumeration of ETEC K88, real-time PCR (qPCR), and Western blotting analysis. Six wells per treatment were used, and the results were representative of 3 independent experiments.
10
Investigating Mechanistic Pathways in Gastric Cancer
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Anti-TAZ (4883S) was purchased from Cell Signaling; anti-CYR61 (SC-13100) from Santa Cruz; anti-actin (RLM3028) from Ruiying Biological. The WWTR1 and luciferase (control) shRNA oligos were synthesized by ShengGong Company. IHC staining S-P kit (KIT-9710) was purchased from MAIXIN Biology Corporation. Atorvastatin calcium was purchased from WuXi Sigma. Transwell dishes were purchased from Corning Inc. Matrigel was purchased from BD Biosciences. The gastric cancer cell line AGS was purchased from the American Type Culture Collection (ATCC).
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