HEK293T cells that were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich or Wako) supplemented with 10% fetal bovine serum (FBS) (Biowest) at 37°C under 5% CO2 in air were seeded on a 35-mm dish (Iwaki) at 5 × 105 cells/well, grown overnight, and transfected with pRc/CMV-mB2R (100 (link)), pcDNA-hTRPV1 (100 (link)), pcDNA-hTRPV1S117A/T371A, pCMV-SPORT6-hADRA2A (DNAFORM, ID 6198830), and/or pGreen-Lantern 1 (101 (link)), which is a humanized green fluorescent protein (GFP) expression vector, using Lipofectamine reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. pcDNA-hTRPV1S117A/T371A was generated from pcDNA-hTRPV1 by site-directed mutagenesis to replace Ser117 and Thr371 with Ala by using a PrimeSTAR Mutagenesis Basal kit (TaKaRa Bio) with the primer sets S117A-S and S117A-AS, and T371A-S and T371-AS (Table S1 ). After incubation for 3 to 4 h, the cells were reseeded on 12-mm coverslips (Matsunami Glass), further incubated for 2 days, and subjected to whole-cell patch-clamp recordings.
35 mm dish
Manufactured by Iwaki
Sourced in Japan
The 35-mm dish is a common laboratory equipment used for cell culture applications. It is a circular, shallow container made of plastic or glass, with a diameter of 35 millimeters. The dish provides a controlled environment for the growth and maintenance of cells, tissues, or small organisms.
Automatically generated - may contain errors
Lab products found in correlation
12 mm coverslips, by Matsunami (1 mentions)
Primestar mutagenesis basal kit, by Takara Bio (1 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dilactate dapi, by Dojindo Laboratories (1 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Poly l lysine (pll), by Matsunami (1 mentions)
Tmr conjugated halotag ligand, by Promega (1 mentions)
4 protocols using 35 mm dish
1
Overexpression of TRPV1 and ADRA2A in HEK293T cells
2
Neutrophil-M. bovis Interaction Assay
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Neutrophils (concentration of 1 × 106 cells suspended in 100 μL RPMI medium with 10% FBS) were seeded onto glass coverslips treated with 0.001% poly-l-lysine (Matsunami glass, Tokyo, Japan) and placed in a 35 mm dish (Iwaki, Shizuoka, Japan). Cells were incubated for 1 h at 37 °C in 5% CO2. Neutrophils were incubated with PMA for 30 min to induce NETs formation (or with PBS for control), and then, 107 CFU octadecyl rhodamine B chloride (Sigma-Aldrich Corp.) labeled live or heat-killed M. bovis (or with PBS for control) were added and incubated for 30 min at 37 °C under a 5% CO2. Neutrophils were washed with PBS and stained with 4,6-diamidino-2-phenylindole, dilactate (DAPI) for 15 min (Dojindo, Tokyo, Japan). Coverslips were washed with PBS, coated with Fluoromount (Diagnostic Biosystems, Pleasanton, CA, USA), and viewed using a fluorescence microscope (Nikon, Tokyo, Japan). Three bovine neutrophil studies were performed individually.
3
Visualizing Cellular Protein Dynamics
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The cultured cells were washed twice with 1 mL of development buffer without Ca2+ or Mg2+ (DB−; 5 mM Na2HPO4 and 5 mM KH2PO4), plated at a density of 5 × 106 cells mL−1 on a 35-mm dish (IWAKI) filled with 1 mL of DB (5 mM Na2HPO4, 5 mM KH2PO4, 2 mM MgSO4, 0.2 mM CaCl2), and incubated for 5–6 at 21 °C. During the last 30 min, 3 nM TMR-conjugated HaloTag ligand (Promega) was added to the cell suspension for observation under TIRFM and 2 μm ligand for confocal microscopy.
4
Fabrication of Porous Fibroin Sponges
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Cocoons (1.6 g) from the Ser1-P1A269 and Ser3-P1A269 lines, as well as nontreated w1-pnd and fibroin fibers (0.1 g) of degummed w1-pnd cocoons, were immersed in 70% and 100% ethanol, air-dried, and dissolved in 12.5 mL of 9M LiBr containing 100 mM Tris-HCl (pH 9.0) for 2 h at 25 °C. Each solution was desalted in 3 L of 1 mM Tris-HCl (pH 9.0) six times using a dialysis membrane with a molecular mass cut-off of 14 kDa (size 36; FUJIFILM Wako Laboratory Chemicals). The dialysate was concentrated by placing the solution in a dialysis membrane to obtain 3 wt% fibroin solution. However, the preparation of the fibroin solution from w1-pnd cocoons was unsuccessful because of the remarkable loss of fibroin through precipitation during dialysis. The successfully obtained fibroin solution was mixed with 1–5% (v/v) methanol in a 35-mm dish (Iwaki), followed by storage overnight at −20 °C to facilitate the formation of porous 3D fibroin sponges. The transparency of sponge samples of the same thickness (4.0 mm) was compared by placing the samples on a black background. Photographs taken 10 cm from the sponges under light conditions were subjected to optical density measurements using ImageJ software (NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/ , accessed on 11 June 2022).
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