Loss of cell viability is due to exposure to fluid shear stress.
A. The viability of PC-3 cells exposed to 10 passages of the FSS assay at 250 µL/s was not significantly different when assessed by three independent techniques: BLI, WST-1 assay, and clonogenic plating (p>0.05 for each pair, Bonferroni’s mulitiple comparison test, minimum of n = 3 for each method). B. Variation in FSS exposure but not cell suspension/preparation conditions significantly affected endpoint viability of PC-3 cells. Conditions tested include altered suspension cell concentration (p>0.05), degree of confluency prior to collection (p>0.05), suspension media (p>0.05), collection technique (p>0.05), and needle length. Only under this final condition, where the time of exposure to FSS was effectively doubled, was a significant difference in endpoint survival noted. (*p<0.01 vs standard 0.5″ needle, Bonferroni’s multiple comparison test. All experiments n = 4 using pump method.) C. FSS survival of PC-3 cells is not affected by changes in pH. PC-3 cells suspended in DMEM/F12, 10% FBS in the presence or absence of 20 mM HEPES (avg. pH at room temperature: 7.3 vs. 7.7, respectively) (p>0.05, Bonferroni’s multiple comparison tests). D. FSS survival of primary cells was not affected by changes in temperature. (p>0.05, Bonferroni’s multiple comparison test, HMEC, n = 2 and PrEC, n = 5 experiments using pump method). E. Response to FSS does not depend on the time cells are held in suspension. Survival is represented as percent viability of non-FSS-treated cells held in suspension for the duration of the assay (p>0.05, one-way ANOVA). F. Loss of viability of PC-3 and primary cells due to detachment-induced cell death during the protocol were not significantly different for up to one hour. Loss of viability due to detachment over the first 30 minutes is insignificant (p>0.05 one-way ANOVA, Bonferroni’s multiple comparison test, n = 5 for each cell line). All error bars = ±SEM. Details on preparations for controls can be found in Methods under Control conditions.
