title: A high content assay to assess cellular fitness.
abstract: A universal process in experimental biology is the use of engineered cells; more often, stably or transiently transfected cells are generated for the purpose. Therefore, it is important that cell health assessment is conducted to check for stress mediated by induction of heat shock proteins (Hsps). For this purpose, we have developed an integrated platform that would enable a direct assessment of transfection efficiency (TE) combined with cellular toxicity and stress response. We make use of automated microscopy and high content analysis to extract from the same well a multiplexed readout to assess and determine optimal chemical transfection conditions. As a proof of concept, we investigated seven commercial reagents, in a matrix of dose and time, to study transfection of an EGFP DNA plasmid into HeLa cells and their consequences on health and fitness; where we scored for cellular proliferation, EGFP positive cells, and induction of Hsp10 and Hsp70 as makers of stress responses. FuGENE HD emerged as the most optimal reagent with no apparent side effects suitable for performing microtiter based miniaturized transfection for both chemical and RNAi screening. In summary, we report on a high content assay method to assess cellular overall fitness upon chemical transfection.
authors: Christophe
authors: C
authors: Antczak
authors: Jeni P
authors: JP
authors: Mahida
authors: Chanpreet
authors: C
authors: Singh
authors: Paul A
authors: PA
authors: Calder
authors: HTS Core Facility, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA. djaballh@mskcc.org.
authors: Hakim
authors: H
authors: Djaballah
mesh_terms: D057205:Automation, Laboratory; D015415:Biomarkers / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008055:Lipids / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010957:Plasmids; D013312:Stress, Physiological / Q000187:drug effects / Q000235:genetics*; D014162:Transfection / Q000379:methods / Q000592:standards*
chemical_list: D015415:Biomarkers; D018835:Chaperonin 10; C411955:FuGene; D018840:HSP70 Heat-Shock Proteins; D008055:Lipids; C086724:Lipofectamine; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
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