Sensitive assay for mycoplasma detection in mammalian cell culture

Abstract

Mycoplasma contamination in mammalian cell cultures is often overlooked yet is a serious issue which can induce a myriad of cellular changes leading to false interpretation of experimental results. Here, we present a simple and sensitive assay to monitor mycoplasma contamination (mycosensor) based on degradation of the Gaussia luciferase reporter in the conditioned medium of cells. This assay proved to be more sensitive as compared to a commercially available bioluminescent assay in detecting mycoplasma contamination in seven different cell lines. The Gaussia luciferase mycosensor assay provides an easy tool to monitor mammalian cell contaminants in a high-throughput fashion.

Figure 1. GAUSSIA luciferase (Gluc) reporter as a measure of mycoplasma contamination in mammalian cell culture

(a–b) conditioned medium from cells expressing Gluc were added on different mycoplasma clean or contaminated cells (1x10⁵ cells plated in a 24-well plate) and the Gluc activity was detected immediately (0 h) and 24 h later either using a luminometer after addition of coelenterazine (a) or by Western blot analysis using anti-Gluc antibody and horse radish peroxidase-conjugated secondary antibody (b). (c) Purified recombinant Gluc (35 ng/ml) was added to mycoplasma positive or negative cells and Gluc activity was detected immediately and 24 h later using a luminometer. (d) A PCR assay which specifically detects the most common strains of mycoplasma was performed on all cell lines used. The smaller band (265–278 b.p., arrow head) results from the amplification of mycoplasma DNA. The larger band (479 b.p., asterisk) is an internal control indicating successful PCR amplification; it is not mycoplasma-specific. (e) Mycoplasma positive or negative cells were treated with Plasmocin for two weeks or left untreated. Three weeks post-treatment, a subculture of these cells was assayed using the Gluc mycoplasma assay. Activity data are presented as the average (from 3 independent experiment) of RLU ratios of 24 h over 0 h ± standard deviation (*P<0.005).

Figure 2. Gluc mycosensor assay optimization and sensitivity comparison

(a) Cell-free conditioned medium from mycoplasma free or contaminated HeLa cells were incubated with recombinant Gluc (35 ng/ml) and assayed immediatey (0 h) and 24 h, 48 h, and 72 h later for Gluc activity using a luminometer. (b) Mycoplasma contaminated and non-contaminated 293T cells were plated in a 24 well plate and incubated with recombinant Gluc. Immediately (time zero) and at different time points, an aliquot of the conditioned medium were assayed for Gluc activity. (c) Recombinant Gluc was added to different amounts of cells plated in a 24 well plate. Immediately and 24 h later, aliquots of conditioned medium were assayed for Gluc activity. (d) Mycoplasma contaminated conditioned medium were diluted (1:200, 1:800, 1:1600) and added to clean 293T cells which were plated the next day in a 24 well plate. Twenty-four h later, recombinant Gluc was added to each well. Immediately and at different time points, aliquots of conditioned medium were assayed for mycoplasma contamination using both the Gluc mycosensor assay and the commercially available MycoAlert® bioluminescent-based assay. The left y-axis corresponds to the Gluc assay results and the right y-axis corresponds to the commercially available assay results. A cutoff ratio of 2 for mycoplasma contamination using the commercially available assay was used as per manufacturer’s instructions.