Take3 micro volume plate reader

The attenuated Salmonella Typhimurium strain BMM50 was a gift from Dr. Stephen McSorely (UC Davis, Davis, CA). BMM50, like previously published S. Typhimurium BRD509 (39 (link)), has a deleted aroA gene and intact riboflavin pathway. Vibrio cholerae serotype 01 Strain El Tor Inaba N16961 was a gift from Dr. Edward Ryan (Massachusetts General Hospital, Boston, MA). All strains were inoculated from single colonies into Luria Bertani (LB) broth shaking 37°C overnight. They were then subcultured and incubated until log phase was reached. Cultures were washed and normalized to 0.1 OD600 using a microplate spectrophotometer (BioTek). Inoculums ranged from 1×10⁶-1×10⁷ colony forming units (CFU) confirmed by plate counts. V. cholerae lysate for ELISAs was prepared as follows. V. cholerae overnight culture was pelleted and washed 3x with cold sterile PBS. Following resuspension, sample was sonicated at 40% power 4 × 1 min intervals, keeping on ice for 1 minute between runs. Sonicated sample was then centrifuged at 15000 rpm × 10 min at 4°C, and the supernatant removed and sterilized using 0.2 μm syringe filter. Protein content was measured using 260/280 absorbance ratio with Take3 Micro-Volume Plate reader (BioTek)

Adult brains were dissected in 1XPBS, transferred to 0.5mL TRIzol reagent (Ambion) and stored at −80°C. Fifteen brains were pooled for each biological replicate. Extraction was done according to TRIzol manufacturer’s specifications. Total RNA was treated with Turbo DNase (Invitrogen) and quantified by absorbance at 260nm on a Take3 Micro-Volume Plate Reader (BioTek). 500ng of RNA from each sample were then used to make cDNA using Superscript III Reverse Transcriptase and random hexamer primers (Invitrogen). 2μL of cDNA was used in reactions using Power SYBR Green Master Mix (Applied Biosystems) and qPCR was performed on a BioRad CFX Connect Real Time System thermocycler. Each sample for each primer set was run in triplicate and a melt profile was observed for each primer set to ensure single product amplifications. PCR cycling parameters were as follows: 96°C - 10min (94°C - 30s, 60°C - 30s. 72°C - 30s) × 40 cycles, 72°C - 1min. fer1HCH and fer2LCH primers were the same as previously used[28 (link)]. Primers for alpha tubulin were as follows: forward: 5’ -ACGTTTGTCAAGCCTCATAGC - 3’; alpha tubulin reverse: 5’-GAGATACATTCACGCATATTGAGTT - 3’. ∆∆Ct calculations were performed using alpha tubulin as the reference to determine fold change of the target transcript.