For the transwell migration assays, Hardy fr.A-D cells were magnetically sorted from BM of wt and Irf4−/− mice as described above (with addition of FITC-conjugated anti-Igµ antibody), and 2 × 105 cells in RPMI (without additives, FCS-free) containing 10 ng/mL rmIL-7 seeded in 50 µL in the top chamber of 96-well 5 µm pore uncoated 96-well transwell plates (HTS transwell® Corning). The bottom chamber was flooded with 200 µL RPMI containing indicated concentrations of rmCXCL12 (Peprotech). After 16 h, inserts were removed, cells in the bottom chamber were collected, counted, and analyzed for B220 surface expression using flow cytometry. The fraction of migrated cells was calculated as n(migrated) × freqB220(migrated)/n(input) × freqB220(input). Normalization to B220+ cells reduced interexperimental differences due to differences in cell purity after magnetic selection. For OP-9 adhesion assays, 5 × 103 OP-9 cells (a gift from Hyun-Dong Chang, DRZF Berlin) were seeded in 96-well microtiter plates 24 h before the assay. On the day of the assay, fr.A-D cells were purified as above and 2 × 105 fr.A-D cells were seeded on top of OP-9 monolayers in RPMI complete + 10 ng/mL rmIL-7. Plates were centrifuged briefly to accelerate cell descension. After 1 h, suspended cells were collected in the supernatant and by washing OP-9 monolayers two times with PBS.
Hts transwell
Manufactured by Corning
Sourced in United States
HTS Transwells are a laboratory equipment product offered by Corning. They are designed for high-throughput screening applications. The core function of HTS Transwells is to facilitate the evaluation of various cell-based assays and experiments that require a permeable support system.
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Lab products found in correlation
13 protocols using hts transwell
1
B Cell Transwell Migration and Adhesion Assays
2
Measuring Epithelial Barrier Function
3
Coculture of Colon Epithelial Cells and Fibroblasts
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The colon epithelial cells–fibroblasts co‐culture was performed using the Corning® HTS Transwell® (Corning). The detailed protocol is provided in Supplemental Methods.
4
Transwell Co-culture of Macrophages and Tumor Cells
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Raw264.7 cells or iBMDMs were co‐cultured with 4T1 tumor cells by using a Transwell culture system with 0.4 μm pore polyester membrane inserts (Corning HTS Transwell, Corning, NY, USA) for 3 days. For the macrophage study, Raw264.7 cells or iBMDMs were seeded in 6‐well plates at 1 × 105 cells/well, and 4T1 cells were seeded at 4 × 105 cells/well in Transwell chambers. For the tumor cell study, 4T1 cells were seeded on plates, while Raw264.7 cells or iBMDMs were seeded in Transwell chambers.
5
Resolvin Modulation of Neutrophil Chemotaxis
6
Co-culture of MCF7 and Th9 Cells
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MCF7 cells were co-cultured with Th9 cells by using a Transwell culture system with 0.4 μm pore polyester membrane inserts (Corning HTS Transwell, Corning, NY, USA) for 3 days. Specifically, MCF7 cells were seeded on plates, while Th9 cells were seeded in Transwell chambers.
7
Endometrial Cancer Cell Invasion Assay
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Cell invasion assays were performed using 96-well HTS transwells (Corning Life Sciences ,Tewksbury ,MA) coated with 0.5-1X BME (Trevigen Inc., Gaithersburg, MD). Endometrial cancer cells or the cells transfected with siRNA hTERT (50,000/well) were starved for 12 hours and then seeded in the upper chambers of the wells in 50 μl FBS-free medium. The lower chambers were filled with 150 μl regular medium or with BIBR. The plate was incubated for 24 hours at 37°C to allow cell invasion into the lower chamber. After washing the upper and lower chambers with PBS once, 100 ul Calcein AM solution was added into lower chamber and incubated at 37°C for 30-60 minutes. The lower chamber plate was measured using a plate reader for reading fluorescence at EX/EM 485/520 nm. The experiments were repeated three times
8
Formulation and Characterization of Br-PLGA Nanoparticles
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Br and PLGA (50:50 (MW 30,000–60,000 Da)) with acid terminated were acquired from Sigma-Aldrich (Darmstadt, Germany). Hanks Balanced Salt Solution (HBSS) was purchased from Bio idea (Iran). HTS Transwells were provided by Corning company (Schnelldorf, Germany). L-α-phosphatidylcholine (95%) (Soy) was obtained from Avanti Company. Other chemical compounds were purchased from Merck (Darmstadt, Germany).
9
Transwell Invasion Assay for SKOV3 and OVCAR5 Cells
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Cell invasion assays were performed using 96-well HTS transwells (Corning Life Sciences, Wilmington, NC) coated with 0.5–1X BME (Trevigen, Gaithersburg, Maryland). Starved (serum-free media for 12 h) SKOV3 and OVCAR5 cells (50,000 cells/well) were seeded for 12 h in the upper chambers of the wells in 50 μl FBS-free medium, and the lower chambers were filled with 150 μl regular medium with different concentrations of everolimus. The plate was incubated for 24 h at 37°C to allow invasion into the lower chamber. After washing the upper and lower chambers with PBS once, 100 μl Calcein AM solution was added into the lower chamber and incubated at 37°C for 30–60 minutes. The lower chamber plate was measured by the plate reader for reading fluorescence at EX/EM 485/520 nM. All experiments were performed in duplicate to assess for consistency of response.
10
Cell Invasion Assay with Simvastatin
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Cell invasion assays were performed using 96-well HTS transwells (Corning Life Sciences, Wilmington, NC) coated with 1X BME (Trevigen, Gaithersburg, Maryland). Starved (serum-free media for 12 h) Hey and SKOV3 cells (50,000 cells/well) were seeded for 12 h in the upper chambers of the wells in 50 μl FBS-free medium, and the lower chambers were filled with 150 μl regular medium with simvastatin. The plate was incubated for 24 h at 37°C to allow invasion into the lower chamber. After washing the upper and lower chambers with PBS once, 100 ul Calcein AM solution was added into the lower 37°C chamber and incubated for 30–60 min. The lower chamber plate was measured by the plate reader for reading fluorescence at EX/EM 485/520 nM. All experiments were performed in duplicate to assess for consistency of response.
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