Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Rhodamine 123 efflux assay

CR Chitra Rawat
RK Rintu Kutum
SK Samiksha Kukal
AS Ankit Srivastava
UD Ujjwal Ranjan Dahiya
SK Suman Kushwaha
SS Sangeeta Sharma
DD Debasis Dash
LS Luciano Saso
AS Achal K. Srivastava
RK Ritushree Kukreti
request Request a Protocol
ask Ask a question
Favorite

After treatment with different chemicals, cells were washed with 1X Phosphate Buffered Saline (PBS). Opti-MEM medium having P-gp substrate, 10 µM rhodamine 123, was added for 1 hour at 37 °C in 5% CO2 to allow its uptake. Media was discarded and cells were washed with ice-cold PBS thrice, followed by incubation for another 1 hour at 37 °C in 5% CO2 in Opti-MEM medium, to allow efflux to occur. Efflux was stopped by washing with ice-cold PBS thrice and cells were lysed with 1% Triton X-100 at 37 °C for 15 min. Fluorescence intensities were measured using Infinite® 200 PRO multimode plate reader (Tecan, Switzerland) with excitation and emission wavelengths of 485 and 538 nm, respectively. Functional activity was measured as absolute fluorescence intensity in cell lysate per mg of total protein.

Copyright and License information: The Author(s) ©2020
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A