phosphate buffered saline d pbs  (Thermo Fisher)

Journal: The Journal of Biological Chemistry

Article Title: Skin-derived α-synuclein strains from PD, DLB, and MSA induce distinct intracellular pathology and neurodegeneration

doi: 10.1016/j.jbc.2025.111005

Figure Lengend Snippet: Intracellular p-αSyn signatures induced by patient skin αSyn strains dictate their seeding activity in vitro. A , schematic of the generation and purification of U251 biosensor cell-derived patient skin or brain αSyn strains. B and C , Western blots showing pathological p-αSyn and total αSyn detected in sarkosyl detergent-insoluble ( B ) and sarkosyl detergent-soluble fraction ( C ) from U251 biosensor cells seeded with patient skin- or brain-amplified αSyn strains. D , quantification of percent p-αSyn normalized to total αSyn in sarkosyl detergent-insoluble fraction from Western blots by densitometric analysis. Data are presented as mean ± SEM, and each dot corresponds to data analyzed using p-αSyn and total αSyn protein band densities in that sample group. N = 6 replicates from biosensor cell-derived skin αSyn strains of 2 cases, N = 3 replicates from biosensor cell-derived brain αSyn strains of 1 case, N = 3 replicates each from biosensor cell-derived αSyn monomer and D-PBS, and N = 6 replicates from biosensor cell-derived rPFF. E – I , RT-QuIC assay ( E–H ) of biosensor cell-derived patient skin or brain αSyn strains showing their in vitro seeding activity. Cutoff was set just below 50,000 RFU using biosensor cell-derived fraction that was initially treated with D-PBS. Quantification of endpoint ThT fluorescence ( I ) at 36-h time-point from RT-QuIC assay showing in vitro seeding activity of patient skin- and brain-amplified αSyn strains following propagation in U251 biosensor cells. Data are presented as mean ± SEM ( dashed lines or bars). Each dot corresponds to data analyzed from N = 10 to 12 replicates from biosensor cell-derived skin αSyn strains of two cases, N = 6 replicates from biosensor cell-derived brain αSyn strains of one case, N = 5, and N = 7 replicates for biosensor cell-derived αSyn monomer and D-PBS, respectively, and N = 7 replicates for RT-QuIC internal control with D-PBS quantified at every 45 min interval for 36 h from two independent experiments. Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple comparison test ( D ) or ordinary two-way ANOVA with Tukey’s multiple comparison test ( E–H ) or Brown–Forsythe and Welch ANOVA with Dunnet’s T3 multiple comparison test ( I ). A p -value < 0.05 was considered significant. Significant differences are indicated by ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. Statistical power is > 0.90 between groups with p -value < 0.05. RT-QuIC, real-time quaking-induced conversion; αSyn, alpha-synuclein; p-αSyn, phosphorylated αSyn at serine 129. ThT, thioflavin T; D-PBS, Dulbecco’s phosphate-buffered saline.

Article Snippet: Briefly, skin tissue was first thawed and then rinsed three times in 700 μl of ice-cold 1X Dulbecco’s phosphate-buffered saline (D-PBS) (Gibco) in 1.5 ml tubes until blood was no longer visible, followed by mincing with a surgical blade into small pieces.

Techniques: Activity Assay, In Vitro, Purification, Derivative Assay, Western Blot, Amplification, Fluorescence, Control, Comparison, Saline