Endogenous plasma resuspension of peripheral blood mononuclear cells prevents preparative-associated stress that modifies polyA-enriched RNA responses to subsequent acute stressors
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
Human peripheral blood mononuclear cells (PBMCs) are used to examine biological processes and disease, when basal variability in cellular activation and splicing is described and unexplained. Using isolation systems that maintained buffy coat cells (PBMCs, platelets) in their own plasma, poly-A enriched RNA-sequencing (RNASeq) detected 42,720 Ensembl gene IDs, including >95% of the top 100 Genotype Tissue Expression Project (GTEx)-expressed genes in lung, colon, heart, skeletal muscle and liver, and 10/17 clinically-actionable genes listed by the Pharmacogenomics Knowledgebase. Transcriptome changes were defined after 1h treatment with 32◦C hypothermia (hsp70 family member change), 10 µmol/L ferric citrate that had no discernible effect, and 100 µg/mL cycloheximide leading to induction of primary response (immediate early) genes including IL1B and TNF. Same-donor PBMCs prepared conventionally using washes then resuspension in serum-supplemented media demonstrated basal upregulation of stress signalling pathway genes that masked and overlapped differential gene expression profiles after 100 µg/L cycloheximide. Plasma-resuspended PBMCs demonstrated minor transcriptome changes after 40 µmol/L ferric citrate, whereas consistent and greater magnitude changes were observed for washed/media- resuspended PBMCs. We conclude that endogenous plasma-maintained PBMCs provide a more robust platform to interrogate acute cellular perturbations trig- gering innate immunity, and that varying susceptibility of PBMCs to preparative stresses is an important component of experimental variability.
Date Issued
2024-11-28
Date Acceptance
2024-10-03
Citation
Cell Stress, 2024, 11 (1), pp.112-124
ISSN
2523-0204
Publisher
Shared Science Publishers OG
Start Page
112
End Page
124
Journal / Book Title
Cell Stress
Volume
11
Issue
1
Copyright Statement
© 2024
Creative Commons License
Endogenous plasma resuspension of peripheral blood mononuclear cells prevents preparative-associated stress that modifies polyA-enriched RNA responses to subsequent acute stressors by Li et al. is licensed under a Creative Commons Attribution 4.0 International License.
Creative Commons License
Endogenous plasma resuspension of peripheral blood mononuclear cells prevents preparative-associated stress that modifies polyA-enriched RNA responses to subsequent acute stressors by Li et al. is licensed under a Creative Commons Attribution 4.0 International License.
License URL
Identifier
https://doi.org/10.15698/cst2024.11.301
Publication Status
Published
Date Publish Online
2024-11-28