The retention time for ethanol (17·84 ± 0·02 min) was determined using a commercial mix (Fuel Ethanol Residual Saccharides Mix; catalogue number 48468-U; Sigma-Aldrich) containing glycerol, glucose, maltotriose, maltose monohydrate, lactic acid, acetic acid, dextrin and ethanol.
Retention time for ethanol (17.84 ± 0.02 minutes), was determined using a commercial mix (Fuel Ethanol Residual Saccharides Mix; catalog number 48468-U; Sigma-Aldrich, St Louis, MO, USA) containing glycerol, glucose, maltotriose, maltose monohydrate, lactic acid, acetic acid, dextrin, and ethanol.
Differences in gene expression resulting from the comparison of the CDCd group with the CONd group were determined using a linear mixed-model in which treatment was included as fixed effect and the sample as random [ 24].
Lymphocyte subset proportions were determined using a commercially available antibody mix containing anti-CD45, anti-CD3, anti-CD4, and anti-CD8 antibodies stantibodies fluorochromestainedaChrome™ CD3-4-8-45, Beckman Coulter).
For this quantification, norvaline was used as the internal standard and the response factor of each amino acid was determined using a 250 pmol/μl standard mix of amino acids.
Relative gene expression was determined using a peqGOLD Hot Start-Mix kit (Peqlab) and a StepOne real-time thermocycler (Applied Biosystems) according to the manufacturer's instructions.
Total RNA was isolated from the midguts of 3 biological replicates with more than 25 flies in each replicates using Trizol as described [ 14]. qPCR was performed following cDNA synthesis and changes in gene expression patterns were determined using a 7500 Fast Real-Time PCR System (andlied BiosysTaqManand TaqMasterster Mix (Applied Biosystems).
Contributions of BrC and internal mixing have been determined using a variety of methods [ 61– 63], however, the multi-wavelength extrapolation methods that have often been applied contain significant uncertainties that should be considered before quantitative attribution is reported [ 62].
The cell lysis was mixed to a homogenized state and the volume was determined using a pipette.
The cell suspension was mixed with trypan blue solution, and the number of live cells was determined using a homocytometer.
