Multiimage light cabinet

Total RNA was extracted from frozen TA, EDL, and soleus muscles using TRIzol reagent (Qiagen, Valencia, CA) and reverse transcribed using oligo(dT) primers and M-MLV reverse transcriptase (Life Technologies), according to the manufacturer's instructions. Primers were as follows: Tmod1 sense, 5′-CAACGCCATGATGAGCAAC-3′; Tmod1 antisense, 5′-CATCGGTAGAACACGTCCAG-3′; Tmod4 sense, 5′-GATGCGGTAGAGATGGAGATG-3′; Tmod4 antisense, 5′-TCTCTT­CTTTTGCTGACGACG-3′; GAPDH sense, 5′-GGGCATCTTGGGCT­ACACT-3′; and GAPDH antisense, 5′-GAGCAATGCCAGCCCCG-3′. PCR was then performed using a Bio-Rad T100 thermal cycler. An initial hold at 95°C for 1 min was followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 20 s. After a final hold at 72°C for 5 min, reaction products were electrophoresed on agarose gels, stained with EtBr, and visualized using a MultiImage Light Cabinet (Alpha Innotech, San Leandro, CA). Tmod1 and Tmod4 band intensities were densitometrically quantified using ImageJ with normalization to GAPDH.

The plasmid (pBluescript-SK+, Fermentas, Waltham, MA, USA) DNA has a supercoiled conformation, but when a single-strand break occurs, it loses that conformation and adapts an open circular conformation. Based on this, the percentage of DNA strand cleavage, as well as the protective activity of food extracts was assessed. Firstly, 2 µl (4 µg/ml) of DNA was mixed with different volumes of sterilised PBS and PM2.5 sample. That way, a gradient of different concentrations of the PM2.5 samples was created. The final volume of the reaction was 10 µl. The samples were incubated for 45 min at 37°C. Subsequently, 3 µl of loading buffer (Bromophenol Blue 0.25% + 30% Glycerol) was mixed to terminate the reaction and the samples were loaded on an 0,8% agarose gel. The samples were ran at 70 V for 55 min. Ethidium bromide was used to stain the gel by suspending it in 12,5 µl of ethidium bromide (10 mg/ml) and 250 ml of distilled water for 30 min. Consequently, the gel was washed with 250 ml distilled water for 20 min. Results were obtained by exposing the gels to UV and capturing a photo using MultiImage Light Cabinet (Alpha Innotech, San Leandro, CA, USA). Finally, we used the Alpha View suite to analyse the photos. When coffee extracts were introduced, the final reaction volume was increased to 13 µl.