Dtx 880 multimode detector

To evaluate PmPV2 fate and internalization Alexa488-labeled PmPV2 and BSA in PBS were used. Three sets of nine mice (females and males) each were gavaged with (i) PBS, (ii) Alexa488-labeled BSA (75 μg) as control, or (iii) Alexa488-labeled PmPV2 (75 μg). At 2, 6 and 8 h post-administration three mice of each treatment were sacrificed and Peyer's patches (PPs) (9 PPs/mice) and the remaining small intestine (SI) were aseptically removed. SI was homogenized with 5 mL of PBS in a Potter type homogenizer OS-40 Pro (DLAB Scientific Inc., Riverside, CA, USA). Single-cell suspensions were prepared (79 (link)) from PPs and washed twice in PBS solution in order to remove extracellular labeled protein. Cell suspensions were resuspended in 5 mL of PBS. Then 100 μL of homogenate or cell suspensions were seeded in 96-well, flat-bottom, black microplate (Corning Inc., Corning, NY, USA) and fluorescence was measured in a microplate reader Multimode Detector DTX-880 (Beckman Coulter Inc., Brea, CA, USA). Arbitrary fluorescence units (A.U.) were corrected by labeling efficiency (effi) and molarity (A.U./effi.mol).

Cells were seeded onto 6-well plate, and then the pcDNA3.1-PKM2 (2 μg/ml), RNA oligonucleotides (NCO, miR-122 mimics, 50 nM) were transfected on the next day. After 24 h post-transfection, the cells were washed with PBS and incubated with fresh DMEM medium with 10% FBS in the absence or presence of DOX (10 μg/ml) for another 24 h. After incubation, the cells were harvested and lysed in 1% NP40 solution, and when the ATP in the lysates combined with the luciferin/luciferase enzyme complex, a reaction which produces light occurs. The relative light units were detected by Multimode Detector (DTX 880, Beckman Coulter).

K562 and K562/ADR cells were placed at an initial density of 5000 cells per well into 96-well plates. After 24 h preincubation, cells were implemented with DOX and matrine treatment at indicated concentrations for 48 h. The viable cells were counted using Cell Counting Kit-8 staining according to the manufacturer's instructions (Dojindo Molecular Technologies, Kumamoto, Japan). Absorbance was read by Multimode Detector DTX 880 (Beckman Coulter, Tokyo, Japan). The concentrations required to inhibit growth by 50% (IC₅₀) were calculated from viability curves using GraphPad Prism 8 (San Diego, California, USA). Reversal fold [19 (link), 20 (link)] values were calculated using the following formula: reversal fold = IC₅₀ of DOX alone/IC₅₀ of DOX in the presence of matrine, to assess the effect of matrine on multidrug resistance. All experiments were performed three times.

Cells were rinsed with ice-cold PBS, and then ice-cold mitochondrial lysis buffer with freshly added phosphatase and protease inhibitors were added (0.2 ml per well of a 12-well plate). Adherent cells were scraped off the dish with a cell scraper and the suspension was transferred into a centrifuge tube and gently rocked for 15–30 min at 4 °C. The lysate was centrifuged at 14 000 × g for 20 min at 4 °C and the supernatant was immediately transferred into fresh pre-chilled micro-centrifuge tubes. The lysate was diluted at 1:4 for BCA assays with a spectrophotometer (Beckman Coulter Multimode Detector DTX880, Beckman Coulter). Finally, the lysate was aliquoted and stored at ⩽−70 °C.

The epithelial barrier function of the intestine was assessed by measuring the serum concentration of FITC-dextran (4 kDa, Sigma-Aldrich) after gavage43 (link). The mice fasted for 4 hours and were given 30 mg/ml of FITC-dextran (0.6 mg/g body weight). Blood was collected 4 hours later by cardiac puncture after CO₂ euthanasia. The supernatant was collected by centrifugation at 3000 rpm for 10 minutes. The fluorescence in serum was measured by using a Multimode Detector DTX880 (Beckman Coulter), and the concentration was determined by comparison with a standard curve of serial-diluted FITC-dextran. For histology, the distal colon was thoroughly flushed with PBS and fixed with 4% paraformaldehyde in PBS at 4 °C overnight, and frozen sections were counterstained with DAPI. Images were examined using fluorescence microscope (ECLPISE 80i EPI, Nikon).

Blood samples (20 μl) were collected periodically from mouse tail veins and centrifuged at 4,000 rpm at 4°C for 20 minutes. Serum samples were assayed for human albumin using a human albumin enzyme-linked immunosorbent assay (ELISA) quantitation kit (Bethyl Laboratories Inc., Montgomery, TX, USA), according to the manufacturer’s instructions. After 6 minutes, reactions were stopped by adding 2 M sulfuric acid, and absorbance was read at 450 nm using a Multimode Detector DTX 880 (Beckman Coulter, Pasadena, CA, USA).