Cytation 5 cell imaging multi mode reader

C57BL/6 wild-type mice were pretreated with high (1 mM) or low (0.1 mM) propionate intratracheally (IT) 2 h prior to IT administration (10⁸ CFU) of luminescent strain of S. aureus (Newman-lux strain generously provided by Alex Horswill, University of Colorado, Denver). IT administration was done under isoflurane anesthesia and with direct visualization. Six hours after infection, mice were live imaged using IVIS^®in vivo imaging system (see below for more details). Lungs were then collected and imaged ex vivo using IVIS^® and luminescence was also measured using Cytation5 cell imaging multi-mode reader (BioTek, Winooski, VT, United States).
Staphylococcus aureus (Newman-lux strain) was grown as follows: after an overnight inoculation in LB broth, serial dilutions of the overnight stock was grown at 37C in a 24-well tissue culture dish with orbital shaking in the Cytation5 cell imaging multi-mode reader (BioTek, Winooski, VT, United States). Every 5 min, an OD reading and a luminescence reading (to verify healthy growth of the luminescence producing strain) were obtained. When the mice were ready for infection, the wells that were at OD 0.3 (mid-log phase) were removed and used for IT infection. Later CFU measurements of these innocula provided the actual CFU count administered to the mice as described above.

The BCA assay was performed in 96-well microplates following the user guide. Briefly, 25 μl of each sample and 200 μl of BCA working reagent were added to each well. The microplate was shaken for 15 seconds and then incubated for 30 min at 37°C using a BioTek^® Cytation 5 Cell Imaging Multi-Mode Reader. Absorbance was measured at 562 nm and protein concentration in samples was determined by interpolation on the bovine serum albumin (BSA) standard curve.
The Bradford assay was performed in 96-well microplates following the user guide. Briefly, 10 μl of each sample and 300 μl of Coomassie reagent were added to each well. After 10 min incubation at room temperature, absorbance was measured at 595 nm using the same BioTek^® Cytation 5 Cell Imaging Multi-Mode Reader and protein concentration in samples was determined by interpolation on the BSA standard curve.
Both colorimetric assays were performed in triplicate on three different days (n = 9). Sample dilutions and standard curves were made with MilliQ water. When SDS was used, both samples and standard curves were diluted with 2% SDS in MilliQ water since both Bradford and BCA kits are detergent resistant. Table 1 summarizes the main advantages and disadvantages of using colorimetric methods for protein determination.