Mouse blood obtained on Day 5 post TAI was processed by centrifugation at 3000 rpm for 15 min to collect the serum. A total of 100 μl of mouse serum was coincubated with 100 μl of tachypleus amebocyte lysate (EC80545, Xiamen Bioendo Technology, Fujian Province, China) at 37°C for 10 min, followed by the addition of 100 μl of chromogenic matrix solution. After coincubation at 37°C for 6 min, hydrochloric acid solution was added to stop the reaction. Azo reagent was finally pipetted into the mixture, the absorbance of which at 545 nm was then determined using a Molecular Devices M2e microplate reader (Sunnyvale, CA). This experiment was conducted in triplicate and repeated three times.
M2e microplate reader
Manufactured by Molecular Devices
Sourced in United States
The M2e microplate reader is a compact and versatile instrument designed for absorbance-based measurements in microplates. It features a broadband xenon flash lamp and a high-precision monochromator to provide accurate and reliable results across a wide range of wavelengths.
Automatically generated - may contain errors
3 protocols using m2e microplate reader
1
Quantifying Endotoxin in Mouse Serum
2
Antibacterial Activity of Peptides
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A virtual colony count assay was used to determine the antibacterial action of the peptides in vitro as previously described [22 (link)]. Briefly, S. aureus (ATCC 25923) and A. baumannii (ATCC 19606) were grown to mid-logarithmic phase and were diluted to 1 × 106 CFU/mL using sodium phosphate buffer (pH = 7.4) containing 1% Müller-Hinton broth (MHB). Peptides were prepared in sterile water at the concentrations of 2.5, 5, 10, 20, 40, and 80 µM. A total of 10 µL of peptides were incubated with 90 µL of microbes at 37 °C for 1 h. Then, another 100 µL of 2 × MHB was added, and bacterial growth was monitored at 600 nm with a M2e microplate reader (Molecular Devices, Silicon Valley, CA, USA) for 12 h. This experiment was conducted in triplicate and repeated twice.
3
Viability Assay of Cells on Scaffolds
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The viability of cells on the scaffolds was analyzed by a live/dead cell staining kit (Dojindo, Japan). After a 24 h coculture, cells attached to the specimens were stained with 500 μL of combination dye for 10 min in the incubator and were then subjected to fluorescence microscopy (LSM780; Carl Zeiss, Germany). At the designated time points (1, 4, and 7 days), the cells were labeled with 5 μg/mL Alexa Fluor 594 phalloidin (Life Tech, US) for 15 min and 4,6-diamidino-2-phenylindole dilactate (DAPI, Life Tech, US) for 5 min. The labeled cells on the scaffolds were observed by laser scanning confocal microscopy (LSM780, ZEISS). High-resolution imaging of Z-stacks was obtained, and confocal Z-stacks of images were processed. Cell cytotoxicity was analyzed with Cell Counting Kit-8 (CCK-8) assays (Dojindo Kagaku, Japan). The solution absorbance was measured at a wavelength of 450 nm with an M2e microplate reader (Molecular Devices, Silicon Valley, CA, US).
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