Depending on the further application, HepG2 cells and HEK293 cells were seeded either on 6-well plates (Techno Plastic Products AG, Trasadingen, Switzerland), glass-bottom dishes (In Vitro Scientific, Sunyvale, CA, USA), or on cover glasses (ThermoFisher Scientific, Loughborough, UK). At next day they were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and an appropriate DNA concentration according to the manufacturer’s instructions. For HEK293 cells, the surfaces were coated with poly-L-lysine (Biochrom AG, Berlin, Germany).
6 well plates
Manufactured by Techno Plastic Products
Sourced in Switzerland
The 6-well plates are a type of cell culture equipment designed for in vitro experiments. Each plate contains six individual wells, providing a convenient platform for conducting parallel experiments or observations. The plates are made of high-quality plastic materials suitable for cell growth and various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Infinite m1000 pro, by Tecan (2 mentions)
Cover glasses, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Harvard Bioscience (1 mentions)
Red cmtpx, by Thermo Fisher Scientific (1 mentions)
Green cmfda, by Thermo Fisher Scientific (1 mentions)
Human il 10 standard abts elisa development kit, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Techno Plastic Products (1 mentions)
Xfect, by Takara Bio (1 mentions)
Hek293t cell line, by American Type Culture Collection (1 mentions)
Hela cell line, by American Type Culture Collection (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
C series human inflammation array c3, by RayBiotech (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Aurum total rna mini kit, by Bio-Rad (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Absolute qpcr sybr green fluorescein mix, by Thermo Fisher Scientific (1 mentions)
9 protocols using 6 well plates
1
Cell Seeding and Transfection Optimization
2
Measuring cAMP in GIST Cell Lines
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GIST882 and GIST48 cells were seeded in 6-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) at a concentration of 250,000 cells/well, 48h before drug treatments. After drug treatments, cell medium was removed and 1ml of HCl 0.1M was added. cAMP was measured by RIA as described [50 (link)].
3
Spheroid Clearance Assay with HPMCs
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Human primary mesothelial cells (HPMCs) were cultured in 6-well plates (Techno Plastic Products, Trasadingen, Switzerland) until >80% confluence was reached. HPMC and CAFs (1:1 ratio) were labeled with CMFDA-green (Molecular Probes, Eugene, OR, USA), washed with PBS, and incubated with fresh cell culture media. Spheroids were prepared as described above but ES2 cells were labelled with CMTPX-red (Molecular Probes) prior to plating in the ultra-low attachment plates. Plates containing labelled cells were incubated at 37 °C, 5% CO2 overnight. Spheroids were added onto the HPMC and CAF monolayer at the microscope and time lapse images were acquired from time zero until 20 h. Clearance ratio was determined by measuring the total area cleared by the spheroid in ImageJ, divided by the size of the spheroid at time zero.
4
SARS-CoV-2 Spike Protein Impacts Interleukin Levels
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The A549-hACE2 cells (2 × 106 cells wells−1, 2 mL well−1) were seeded in 6-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) and kept overnight for attachment. Cells were incubated with Dt in DMSO at the concentration of 0.1, 1, 10, and 100 ng mL−1, with or without recombinant human coronavirus SARS-CoV-2 spike glycoprotein RBD (His tag) (cat. No. ab275986, Abcam, Darmstadt, Germany). After the 24 h-lasting incubation, cell media were collected and the evaluation of the interleukins IL-6 and IL-10 concentrations was carried out through sandwich ELISA. Human IL-6 Standard ABTS ELISA Development Kit (cat. No. 900-K16, PeproTech, London, UK) and Human IL-10 Standard ABTS ELISA Development Kit (cat. No. 900-K21, PeproTech, London, UK) were used according to the manufacturer’s protocol. The absorbance was measured at 405 nm (with wavelength correction set at 650 nm) using the Microplate Reader: Infinite® M1000 PRO (TECAN, Männedorf, Switzerland).
5
Cell Transfection Protocol for HepG2 and MCF-7 Cells
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HepG2 and MCF-7∆AHR cells were seeded either on 10-cm dishes, 6-well plates (Techno Plastic Products AG, Trasadingen, Switzerland), 96-well plates (Techno Plastic Products AG, Trasadingen, Switzerland), or on glass bottom dishes (In VitroScientific, Sunyvale, CA, USA). On the next day, cells on glass bottom dishes were transfected by using Xfect (Takara Bio Europe SAS, Saint-Germain-en-Laye, France) and an appropriate DNA amount according to the manufacturer’s instructions. Cells on 10-cm dishes or on multiwell plates were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and an appropriate DNA amount as stated by the manufacturer’s instructions. After 4 h incubation, transfection medium was removed and replaced by new RPMI or DMEM medium, respectively.
6
Cell Culture: HEK293T and HeLa
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The HEK293T cell line (ATCC) was used for all experiments in this study; the HeLa cell line (ATCC) was used only for the T3 ligation assay. The source for commercial HEK293T as well as HeLa cell line is reported to be female. Cells were grown in Dulbecco’s modified Eagle’s Medium (DMEM) high glucose medium supplemented with 10% fetal bovine serum (FBS) at 37.0°C with 5.0% CO2 concentration. Cells were seeded into 6-well plates (Techno Plastic Products) to 2.5 mL of medium per well and grown 24 h prior to transfection at approximately 40–50% confluency.
7
Antibody Array Analysis of Inflammation Proteins
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The regulation of the proteins involved in the inflammation process was investigated through an antibody array performed using RayBiotech® C-Series Human Inflammation Array C3 (code: AAH-INF-3, RayBiotech, Peachtree Corners, GA, USA). For this aim, U937 cells (2 × 106 cells−1) were seeded in 6-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland). For the detection of proteins, cells were treated for 24 h at a concentration of Bbm of 10 μg mL−1. After incubation, cell medium was collected from control and treated cells. Protein concentration and purity were assessed using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 1 mL of sample was used to perform the antibody array, according to the manufacturer’s protocol. Blots were analyzed using ImageLab software (Bio-Rad, Hercules, CA, USA) and results are shown in terms of relative expression.
8
Assessing Effect of Bb on IL-6 Release
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In order to assess the effect of Bbm on the release of IL-6, U937 cells (2 × 106 cells) were seeded in 6-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) and kept overnight for attachment. U937 cells were treated with 10 μg mL−1 of Bbm and, after 24 h, media were collected from control (no Bbm) and treated cells. After incubation, cell media were collected and used to evaluate the release of cytokines, by sandwich ELISA detection, using Human IL-6 Standard ABTS ELISA Development Kit (cat. No. 900-K16, PeproTech, London, UK), according to manufacturer’s protocol. The absorbance was measured at 405 nm (with wavelength correction set at 650 nm) using Microplate Reader: Infinite® M1000 PRO (Ex: 320 nm, Em: 420 nm, TECAN, Männedorf, Switzerland). IL-6 levels were expressed in ng mL−1 of medium (using IL-6 standard curve).
9
Selenite Regulation of SELENOP in HepG2 Cells
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HepG2 cells were seeded at 2×106 cells per well in 6-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) and cultivated at 37 °C and 5% CO2 in DMEM/F12 (Gibco, Thermo Fisher Scientific Inc.) +10% FCS (Biochrom GmbH, Berlin, Germany). The medium was exchanged at 80% confluency after 24 h, and cells were further cultivated for 24 h either in serum-free DMEM/F12 or DMEM/F12 containing 10% FCS. On the third day, a concentration range of sodium selenite (1 nM–10 µM, f.c.) was prepared in serum-free or FCS-containing medium, and treatment was started for additional 24 h by exchanging the medium. Then, cells were harvested, total RNA was isolated using Aurum™ Total RNA Mini Kit (Bio-Rad Laboratories, Inc, Hercules, California, U.S.A.) and first-strand cDNA was synthesized using iScript™ cDNA Synthesis Kit (BioRad). Relative transcript levels of SELENOP (forward primer: 5´-TATGATAGATGTGGCCGTCTTG−3´; reverse primer: 5´-TGTGATGATGCTCATGATGGTA-3´) were determined using the ABsolute QPCR SYBR Green Fluorescein Mix (Thermo Fisher Scientific) and beta actin (ACTB; forward primer: 5´-CACCACACCTTCTACAATGAGC−3´; reverse primer: 5´-CAGAGGCGTACAGGGATAGC-3´) as housekeeper for normalization.
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