Chapter 4 96 Dithiothreitol (DTT, 200 mg/mL) is a general reagent reducing THP polymerization,16,35 though excess DTT also breaks the disulfide bond in other molecules, such as present on antibodies.36 The final concentration of DTT should be carefully considered because there are no washing steps in our protocol. Due to the small sample volume (500 µL) used for each test, reducing the volume of DTT makes it hard to resuspend the urine pellet. Therefore, we opted to reduce the concentration of DTT: we used 25 mg/mL and 200 mg/mL DTT to examine the effect of DTT on uEV detection using IFCM. Compared to the non-DTT group, 25 mg/mL of DTT did not affect CD63+ uEVs concentration in HC urine samples, whereas 200 mg/mL of DTT caused a 9.5 ± 5.2% decline of detected uEV amounts (p = 0.0431; Figure 7D). In KTR urine, 25 mg/mL DTT elevated 12.4 ± 10.4% in CD63+ uEV concentration compared to the non-DTT usage (p = 0.0367; Figure 7D). However, 200 mg/mL DTT showed a 13.83 ± 3.7% decrease in uEV numbers compared to the non-DTT group (p = 0.0109; Figure 7D) in KTR urine. Hence, 25 mg/mL of DTT was used in our developed protocol for applications in patient urine with high levels of THP.
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