Recognition of RNA by receptors of the innate immune system is regulated by various posttranslational modifications. Different single 2′-O-ribose (2′-O-) methylations have been shown to convert TLR7/TLR8 ligands into specific TLR8 ligands, so we investigated whether the position of 2′-O-methylation is crucial for its function. To this end, we designed different 2′-O-methylated RNA oligoribonucleotides (ORN), investigating their immune activity in various cell systems and analyzing degradation under RNase T2 treatment. We found that the 18S rRNA-derived TLR7/8 ligand, RNA63, was differentially digested as a result of 2′-O-methylation, leading to variations in TLR8 and TLR7 inhibition. The suitability of certain 2′-O-methylated RNA63 derivatives as TLR8 agonists was further demonstrated by the fact that other RNA sequences were only weak TLR8 agonists. We were thus able to identify specific 2′-O-methylated RNA derivatives as optimal TLR8 ligands.
Background
In recent times, the study of metabolic pathways has become inevitable and predominant for a variety of research fields as cancer biology and immunology. L-lactate as a product of anaerobic glycolysis has shown to be an important indicator of the cellular metabolic status and can be associated with diverse cellular effects. For this reason, L-lactate assay kits are of high demand when metabolic effects need to be considered. Nevertheless, commercially available kits are not affordable if multiple samples must be evaluated.
Principal finding
In this work, we develop an easy and cost-effective colorimetric assay for quantification of L-lactate suitable for cells with low or high L-lactate production based on LDH activity and suitable for 96 well-plate format. Using different metabolic regulators, we demonstrate the capacity of the assay to detect and quantify L-lactate from the supernatant of HeLa cancer cell line. Furthermore, we validate the assay against a commercially available kit by demonstrating no significant difference between both assays. Finally, we show that the assay is capable of quantifying L-lactate in primary cells such as hPBMCs that were stimulated with toll-like receptor ligands and treated with different metabolic regulators.
Conclusion
We herein present an easy custom assay that is suitable for cells with low and high L-lactate production at very low cost compared to commercially available kits. These advantages of the custom assay can simplify the research in the field of metabolism and related fields.
As a product of anaerobic glycolysis L-lactate has shown to be an important indicator of the cellular metabolic status and can be associated with diverse cellular effects. For this reason, L-lactate assay kits are in high demand when metabolic effects need to be examined. Nevertheless, commercially available kits are not affordable if multiple samples must be evaluated. This protocol uses LDH activity to determine the concentration of L-lactate as a product of metabolism in cellular supernatant and is suitable for 96 well-plate format. The protocol provides repeatable results and exhibits an easy and cost-effective alternative for any lab with a microplate absorbance reader capable of measuring millimolar amount of L-lactate in supernatant.
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