Overexpression of TRPV1 and ADRA2A in HEK293T cells

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-hTRPV1_S117A/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-hTRPV1_S117A/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.

Free full text: Click here

Hiramatsu Y., Suzuki K., Nishida T., Onoda N., Satoh T., Akira S., Ikawa M., Ikeda H., Kamei J., Derouiche S., Tominaga M, & Horiguchi Y. (2022). The Mechanism of Pertussis Cough Revealed by the Mouse-Coughing Model. mBio, 13(2), e03197-21.

Publication 2022

35-mm dish Lipofectamine reagent PrimeSTAR Mutagenesis Basal kit 12-mm coverslips

Cells Dish Eagle Fetal bovine serum Green fluorescent protein Lipofectamine Mutagenesis Primer Site directed mutagenesis Vector

Corresponding Organization : Osaka University

Other organizations : Kyoto Biken Laboratories (Japan), Osaka International University, Hoshi University, Juntendo University, National Institute for Physiological Sciences, National Institutes of Natural Sciences

Top 5 similar protocols

Variable analysis

independent variables

PRc/CMV-mB2R (100 ng)
PcDNA-hTRPV1 (100 ng)
PcDNA-hTRPV1(S117A/T371A)
PCMV-SPORT6-hADRA2A
PGreen-Lantern 1 (101 ng)

dependent variables

Whole-cell patch-clamp recordings

control variables

HEK293T cells
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO2 in air
Cells seeded on a 35-mm dish at 5 × 105 cells/well, grown overnight
Transfection using Lipofectamine reagent

controls

Not explicitly mentioned
Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!