Gene Expression Analysis of Sorted Cells by RNA-seq in Drosophila Intestine

A feedback amplification loop between stem cells and their progeny promotes tissue regeneration and tumorigenesis

“…no. E0258), as described in detail at Bio-protocol ( Chen et al, 2016 ). Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 50 ng of total RNA for the construction of sequencing libraries.…”

Hazard Assessment of the Effects of Acute and Chronic Exposure to Permethrin, Copper Hydroxide, Acephate, and Validamycin Nanopesticides on the Physiology of Drosophila: Novel Insights into the Cellular Internalization and Biological Effects

“…Internalization of nanopesticides within the intestinal barrier was assessed in 3rd-instar Drosophila larvae (exposed for four days), which were collected, washed carefully, dissected in phosphate buffer (PB; 0.1 M, pH 7.4), and manipulated in line with our previously described protocol [ 88 , 157 , 158 , 159 ]. Firstly, 0.15 M (1X) PBS (137 mM NaCl (sodium chloride), 2.7 mM KCl (potassium chloride), 4.3 mM Na 2 HPO 4 (disodium hydrogen phosphate), 1.4 mM KH 2 PO 4 (potassium dihydrogen phosphate)) was prepared, and then 0.15 M PBS was diluted with distilled water at a ratio of 2:1 to obtain 0.1 M PBS.…”

“…Midguts isolated from the larvae were fixed in a fixative solution for 2 h at +4 °C. Midgut dissection performed in Drosophila specimens is shown in the literature [ 158 , 159 , 160 ]. The following methods were used to examine the isolated midguts through TEM: For fixation, the samples were kept in 4% glutaraldehyde (prepared in 0.1 M Sorensen phosphate buffer) at +4 °C for 2 h. They were washed for 10 min (3 repetitions) on a rotator in 0.1 M Sorensen phosphate buffer at room temperature.…”

“…Intracellular ROS generation was assessed by dichloro-dihydro-fluorescein diacetate (DCFH-DA) screening in hemocytes and midgut cells from Drosophila larvae once they had been exposed to the chemicals [ 161 ]; the procedure is described in our previously published papers [ 88 , 138 , 157 , 158 , 159 ]. To summarize, after their collection from the flies, the hemocytes and midgut cells were exposed to 5 mM DCFH-DA for 30 min at 24 °C.…”

“…DNA breakage was assessed by comet assays, where the hemocytes collected from the Drosophila larvae were used as target cells. The procedures described in previously published works in the literature were followed at every stage [ 75 , 138 , 159 , 161 , 163 , 164 ]. Third-instar larvae (72 ± 4 h old) were transferred into plastic vials previously filled with 4 g of Drosophila instant food culture that was pre-wetted with the following concentrations: control (distilled water) at 0, and study groups at different concentrations of permethrin nanopesticides (0.01, 0.1, 1, and 2.5 mM), permethrin (0.01, 0.03, 0.06, and 0.1 mM), Cu(OH) 2 nanopesticides and CuSO 4 ·5H 2 O (0.01, 0.1, 1, and 5 mM), acephate nanopesticides and acephate (0.01, 0.1, 1, and 5 mM), and validamycin nanopesticides and validamycin (0.01, 0.1, 1, and 2.5 mM) dispersions, for 24 ± 2 h. Clean distilled water was used as a negative control [ 76 ], while EMS (4 mM) [ 159 , 161 ] was used as positive control.…”