2 research outputs found
Toxic effects of trace phenol/guanidine isothiocyanate (P/GI) on cells cultured nearby in covered 96-well plates
Background: A mixture of phenol and guanidine isothiocyanate (“P/GI”, the principal components of TRIzol™ and similar products) is routinely used to isolate RNA, DNA, and proteins from a single specimen. In time-course experiments of cells grown in tissue culture, replicate wells are often harvested sequentially and compared, with the assumption that in-well lysis and complete aspiration of P/GI has no effect on continuing cultures in nearby wells. Methods: To test this assumption, we investigated morphology and function of RAW 264.7 cells (an immortalized mouse macrophage cell line) cultured in covered 96-well plates for 4, 8, or 24 h at varying distances from a single control well or a well into which P/GI had been deposited and immediately aspirated completely. Results: Time- and distance-dependent disruptions resulting from proximity to a single well containing trace residual P/GI were seen in cell morphology (blebbing, cytoplasmic disruption, and accumulation of intracellular vesicles), cell function (pH of culture medium), and expression of genes related to inflammation (Tnfα) and autophagy (Lc3b). There was no transcriptional change in the anti-apoptotic gene Mcl1, nor the pro-apoptotic gene Hrk, nor in P/GI-unexposed control cultures. LPS-stimulated cells incubated near P/GI had lower expression of the cytokine Il6. These effects were seen as early as 4 h of exposure and at a distance of up to 3 well units from the P/GI-exposed well. Conclusions: Exposure to trace residual quantities of P/GI in covered tissue culture plates leads to substantial disruption of cell morphology and function in as little as 4 h, possibly through induction of autophagy but not apoptosis. This phenomenon should be considered when planning time-course experiments in multi-well covered tissue culture plates.</p
Recommended from our members
Maternal plasma and salivary anelloviruses in pregnancy and preterm birth
Introduction: Human anelloviruses, including torque teno virus (TTV) and torque teno mini virus (TTMV), are ubiquitous in the general population and have no known pathogenicity. We investigated the prevalence and viral load of TTV and TTMV in plasma and saliva over pregnancy, and assessed their association with spontaneous or medically indicated preterm birth. Methods: This is a secondary analysis of the Measurement of Maternal Stress (MOMS) study, which recruited 744 individuals with singleton pregnancies from 4 US sites (Chicago, Pittsburgh, San Antonio, and rural Pennsylvania). Baseline outpatient visits took place in the second trimester (between 12′0 and 20′6/7  weeks’ gestation), and follow-up visits in the third trimester (between 32′0 and 35′6/7  weeks’ gestation). In a case-control study design, participants who delivered preterm ( Results: TTV was detected in plasma from 81% (second trimester) and 77% (third trimester) of participants, and in saliva from 64 and 60%. Corresponding detection rates for TTMV were 59 and 41% in plasma, and 35 and 24% in saliva. TTV and TTMV concentrations were similar between matched plasma and saliva samples. TTV prevalence and concentrations were not significantly different between groups (sPTB, iPTB, and controls). However, plasma TTMV in the third trimester was associated with sPTB and earlier gestational age at delivery. The iPTB group was not different from either the sPTB or the control group. In saliva, concentrations of TTV and TTMV were similar among the three groups. Both TTV and TTMV were more prevalent with increasing parity and were more common in Black and Hispanic participants compared to non-Hispanic White participants. Conclusion: Anellovirus presence (specifically, TTMV) in the third trimester may be associated with preterm birth. Whether this association is causative remains to be determined.</p