2+KG1a-exosomes were added and were incubated overnight, rotating at 4°C. Next, three washes using PBS with the respective Caor EDTA concentration took place, and finally, the E-selectin cargo was eluted. For the elution, 8M urea was used—Enough to cover all the beads- and a 5 min incubation at room temperature took place. Afterward, NuPAGELDS Sample Buffer was added in equal volume to the urea, and was allowed to incubate for another 5 minutes at room temperature.
FTVI-treated and buffer-treated K562-derived exosomes were incubated with Dynabeads coupled with rE-selectin-IgG in the presence of 2 mM Caprior to elution using 50 mM EDTA for 5 min . The downstream processing was the same as for the KG1a-derived exosomes. The methodology that was used for the pulldown of the exosomes derived from the primary cells, was the same as the one for the KG1a-derived exosomes.: Exosomes were stained with the Vybrant DiD Cell-Labeling Solution by Thermo Fisher.
Commercially available recombinant human E-selectin was deposited into a µ-Slide VI 0.1 uncoated microfluidics chamber overnight at 4°C. The chamber was washed using PBS. Exosomes stained with DiD were resuspended in either 2 mM of Ca) and then incubated for 30 min at room temperature. The chamber was washed four times using PBS and then imaged.
Sample preparation- The rE-selectin was labeled using the Monolith NT His-Tag Labeling Kit RED-tris-NTA . To assess binding detection , the final concentration of the rE-selectin was 1 nM, and the final concentration of the exosomes was 11 nM. The mixture contained 0.05% Tween 20 and 2 mM Ca. After mixing, the samples were centrifuged for 10 min at 10,000 rpm and finally loaded onto Premium Capillaries . The samples were run on the Monolith NT.115Pico .
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