Human cell lines used in the in vitro studies included preferred niches of colonization/infection of S. pyogenes from the respiratory track: Detroit 562 (ATCC® CCL138™), a cell line derived from the metastatic site of pharynx carcinoma, primary Bronchial/Tracheal Epithelial Cells (BTEC) (ATCC® PCS300010™), and A549 (ATCC® CCL185™), a cell line derived from a human adenocarcinoma of the alveolar basal epithelial cells. All cell lines were purchased from the American Type Culture Collection (ATCC) (www.atcc.org ) and cultured according to the manufacturer′s specifications. Detroit 562 and A549 cultures were maintained in Dulbecco's modified eagle medium (DMEM, ThermoFisher Scientific) supplemented with 10% (v/v) Fetal Bovine serum (ThermoFisher Scientific), and a mixture of 100 U/ml penicillin and 100 μg/ml streptomycin (ThermoFisher Scientific). BTEC were maintained in Airway Epithelial Cell Basal medium (ATCC) supplemented with Bronchial epithelial cell growth kit (ATCC), 33 μmol/l Phenol Red (Sigma) and a mixture of 100 U/ml penicillin and 100 μg/ml streptomycin (ThermoFisher Scientific). For bacterial internalization and adherence assays, human cells were seeded in a 96‐well culture plate (Sigma‐Aldrich Co. LLC, St. Louis, United States of America) at a density of 3 × 104 cells/well and incubated for 24 hours at 37°C, 5% (v/v) CO2 and 99% (v/v) relative humidity.
96 well culture plate
Manufactured by Merck Group
Sourced in United States
The 96-well culture plate is a laboratory equipment used for cell culture and assays. It provides a standardized multi-well format to cultivate and analyze various cell types in a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Ccl 185, by American Type Culture Collection (1 mentions)
Pcs 300 010, by American Type Culture Collection (1 mentions)
Detroit 562, by American Type Culture Collection (1 mentions)
Phenol red, by Merck Group (1 mentions)
Bronchial epithelial cell growth kit, by American Type Culture Collection (1 mentions)
Detroit 562, by Thermo Fisher Scientific (1 mentions)
Arvo mx, by PerkinElmer (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Six well plates, by Merck Group (1 mentions)
Rhvegf, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Co2 incubator, by Sanyo (1 mentions)
4 protocols using 96 well culture plate
1
In Vitro Evaluation of S. pyogenes Infection
2
Investigating c-Met Inhibitor Efficacy
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Cells (3×105/well) were seeded into 96 well culture plate (Sigma). One day post seeding, cells were treated with c-Met inhibitors for 3, 5, and 7 days. Cell viability was detected by WST-8 assay (Cell Counting Kit-8 (DOJINDO)). The formazan dye formed was measured by ARVOTM MX (PerkinElmer) at 450 nm. Numerical values of test groups are shown with respect to 0.02% DMSO treated group.
3
VEGF-Induced Endothelial Progenitor Cell Proliferation
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The proliferation assay was performed as previously described [12 (link)]. Briefly, EPCs were digested with 0.25% trypsin (Sagene) and then seeded in 96-well culture plates (Sigma). After overnight culture in phenol red-free DMEM, cells were incubated with 100 ng/mL rhVEGF (ProSpec, East Brunswick, NJ, USA) for 24 hours. Before the optical density measurement (562 nm, Tecan, Zurich, Switzerland) was performed, EPCs were supplemented with 10 μL MTT (5 g/L, Gibco) for another six hours and 200 μL DMSO for 10 minutes.
Mitogenic activity was further evaluated by cell counting. EPCs were seeded in six-well plates (Sigma) at a density of 5 × 105 cells per well in phenol red-free DMEM overnight, and then exposed to 100 ng/mL rhVEGF for another 24 hours.
Mitogenic activity was further evaluated by cell counting. EPCs were seeded in six-well plates (Sigma) at a density of 5 × 105 cells per well in phenol red-free DMEM overnight, and then exposed to 100 ng/mL rhVEGF for another 24 hours.
4
Monocyte Cytokine Production on Bionanofilms
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Monocytes (MNs) were isolated in hypertonic density gradient of Ficoll-verografin, according to the Recalde H. [14 (link)]. The isolated cells were suspended in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal serum (Thermo Fisher Scientific, Waltham, MA, USA) and the cell concentration was adjusted to 2 × 106 cells/mL. In 96-well culture plates (Sigma-Aldrich, St. Louis, MO, USA) disks of bionanofilms were placed on the wells bottom and 100μL of DMEM medium and 50μLof cell suspension per well were added (105 cells in each well). A culture plastic (CP) served as a control. Cells were cultured in a CO2-incubator (SANYO Electric Co., Ltd., Osaka, Japan) for 6 days, whereupon cultural filtrates were collected. In cultural filtrates, the content of interleukin-6 (IL-6) and interleukin-10 (IL-10) was determined by enzyme immunoassay (Human IL-10, Human IL-6 ELISA Kit, Biolegend, San Diego, CA, USA).
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