Infinite m200 microplate reader

The cytotoxicity of the compounds against tumor cell lines was analyzed using colorimetric MTT-test with the detection using plate Infinite M200 microplate reader (Tecan, Switzerland). This test is used to assess the metabolic activity of cells. 48 h after the addition of compounds, the medium was removed from each well and 100 µL of fresh medium with MTT reagent was added until the final MTT concentration of 0.5 mg/mL. Then the plates were placed in a CO₂ incubator at 37 °C for 4 h. After that, 150 mL of dimethyl sulfoxide (DMSO) solution was added to the wells and incubated for another 15 min on a shaker. The optical density was measured at a wavelength of 590 nm using Infinite M200 microplate reader (Tecan, Männedorf, Switzerland). Cell viability was calculated as a relative value, taking the optical density values in the control wells as 100%. Data processing was carried out in the GraphPad Prism 8.

To scrutinize the role of miR-2909 on mitochondrial function, various parameters include quantification of ATP levels, mitochondrial cytochrome c oxidase activity and secreted lactate concentrations in human PBMCs transfected with either miR-2909 expression vector or control vector after 48h post-transfection using their respective kits. Cells were lysed using cell lysis buffer supplied by Biovision. ATP levels were quantified using ATP kit (ATP colorimetric/fluorometric assay kit, BioVision) according to the manufacturer's instructions. ATP values were normalized to protein concentrations quantified with the Bradford assay protocol [19 ]. Mitochondrial cytochrome c oxidase activity was measured using the cytochrome c oxidase assay kit supplied by Biovision using automatic plate reader (Infinite M200 Microplate Reader; Tecan).Bradford assay was used to quantify the protein concentration. Secreted lactate concentrations in cell culture media of control and miR-2909 transfected PBMCs were determined by using L-Lactate Assay Kit (Cayman Chemical, Ann Arbor, Michigan, USA). The fluorescent product was analyzed with an excitation wavelength of 530–540 nm and an emission wavelength of 585–595 nm using automatic plate reader (Infinite M200 Microplate Reader; Tecan). Lactate values were normalized to protein concentrations quantified with the Bradford assay protocol [19 ].

Intracellular ATP levels of E. coli B2 were determined using an Enhanced ATP Assay Kit (Beyotime, China). E. coli B2 suspension was incubated with thymine (0, 5, and 10 mM) for 1 h. After incubation, bacterial cells were harvested and lysed with lysozyme, and the supernatant was prepared for ATP levels measurement. Detecting solution was added to a 96-well plate and incubated at room temperature for 5 min. Subsequently, the supernatants were added to the wells and its luminescence was measured by Infinite M200 Microplate reader (Tecan). Total ATP levels were calculated from the luminescence signals accordingly.
2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) were added into E. coli B2 suspension (Liu et al., 2020a (link)). After incubated at 37°C for 30 min, 190 μL of probe-labeled bacterial cells were added to a 96-well plate and 10 μL of ciprofloxacin (0, 1, 5, and 10-fold MIC) without or with thymine (10 mM) were added. After incubation at 37°C for 1 h, fluorescence units were immediately measured with the excitation wavelength at 488 nm and emission wavelength at 525 nm using an Infinite M200 Microplate reader (Tecan).

Cell viability of CaCO2 cells treated with MakA for the indicated time (24 h or 48 h) was quantified by MTS (Promega) or Presto Blue cell viability assays, according to manufacturer’s instructions. MTS absorbance and Presto Blue fluorescence was measured on an Infinite M200 microplate reader (Tecan). Data was normalized to the vehicle treated cells and expressed as a percentage of the control. The cell toxicity of HCT8 cells treated with bacterial supernatants was quantified by measuring propidium iodide fluorescence on an Infinite M200 microplate reader (Tecan). Data was normalized against cells treated with 0.1% triton X-100.

B. cereus strains were grown overnight at 37°C under low iron conditions by inoculating strains in LB medium supplemented with 200 µM 2,2′-dipyridyl. Overnight cultures were inoculated into a new LB medium +200 µM 2,2′-dipyridyl at a final OD of 0.01. Bacteria from mid-log phase culture were washed twice in LB medium containing 600 µM 2,2′-dipyridyl, then inoculated to a final optical density (OD) of about 0.005 into LB medium or LB+600 µM 2,2′-dipyridyl +0.3 µM HoSF supplemented or not with 5 µM Enterobactin (Sigma-Aldrich). Stock solution of ferritin was prechelated in 2 mM 2,2′-dipyridyl for two hours in order to eliminate the free iron. B. cereus cells were grown at 37°C in 96-wells microtiter plate under continuous shaking. The OD was measured at 600 nm every hour over 16 hours using a TECAN Infinite M200 Microplate Reader (TECAN Group). The assays were repeated at least three times.

Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Shanghai, China). MLO-Y4 osteocytes were seeded at a density of 3 × 10³ cells on 96-well plates, and cultured for 24 h. The cells were treated with 0 (control), 0.1 mg/mL, or 1.0 mg/mL of Ti particles. Cell proliferation was examined at 24 h, 48 h, and 72 h after treatment with Ti particles. Briefly, α-MEM (100 μL) with 10 μL of CCK-8 was added to each pre-cultured well, and then the plates were incubated for 2 h at 37 °C. The absorbance at a wavelength of 450 nm was determined using a TECAN Infinite M200 microplate reader (Tecan Group, Mannedorf, Switzerland).