Antibiotic antimycotic mix

Corneal organ cultures were established as described in details⁵⁸ and were maintained in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific) with 1X insulin-transferrin-selenite (Sigma-Aldrich), 1X non-essential amino acids, and 1X antibiotic/antimycotic mix (Thermo Fisher Scientific).

S2R+ cells (Drosophila Genomics Resource Center, NIH Grant 2P40OD010949) are described herein as S2 cells. The cells were cultivated at 27°C in Schneider’s Drosophila medium with 5% fetal calf serum and a 1% antibiotic-antimycotic mix (all from Thermo Fisher Scientific) in six-well plates for transfection and in T25 flasks for subculturing. HEK-293T cells were grown in RPMI-1640 GlutaMAX medium with 5% fetal calf serum and a 1% antibiotic-antimycotic mix in a 5% CO₂ atmosphere at 37°C with a relative humidity of ~93%. Cells were transferred to six-well plates in Opti-MEM for transfections using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. To induce expression of constructs using the pMT plasmids, S2 cells were treated with 5 mM CuSO₄.

Human primary LECs were transfected with 50 nM hsa-miR-146a-5p pre-miR miRNA precursor or anti-miR miRNA inhibitor, or their respective negative controls using Lipofectamine RNAiMAX transfection agent (all from Thermo Fisher Scientific) following the manufacturer’s instructions. Cells were harvested 3 days post-transfection for protein and RNA analysis or processed for immunostaining. Corneal organ cultures [10 (link)] were maintained in Dulbecco’s Modified Eagle’s Medium with 1X insulin-transferrin-selenite (Sigma-Aldrich, St Louis, MO, USA) and 1X antibiotic/antimycotic mix (Thermo Fisher Scientific).

Mice were housed and used in accordance with the Home Office Animal Scientific Procedures Act (ASPA), 1986 under project licence P98A03BF9. The upper cervical spinal cord-lower brainstem tissue was dissected from newborn (P0) male and female mice (n = 6 mice/each culture; C57BL/6 J, RRID: IMSR_JAX:000664 l; provided by Dr Andrea Loreto). The tissue was physically dissociated into small pieces, followed by enzymatic digestion with 0.25% trypsin and 0.2% collagenase in a mechanical dissociator (Gentle MACS Octo dissociator, Milteny Biotec) at 37 °C for 30 minutes. The obtained single cell suspension was plated in poly-D-Lysine coated T75 flasks and kept in DMEM supplemented with 10% foetal bovine serum (FBS) and 1% antibiotic-antimycotic mix (Thermo Fisher Scientific, 15240096). At day 7, top dwelling progenitor cells and microglia were removed by overnight shaking at 100 rpm; then the culture was differentially passaged using 0.0025% trypsin to remove microglia. Finally, 0.025% trypsin was applied for 2-3 minutes to detach astrocytes for replating, while fibroblast mostly remained attached. Further purification was carried out by GLAST antibody-tagged magnetic beads by published methods^{14 (link)}, which resulted in high-purity astrocyte cultures ( > 98%).

Human embryonic kidney 293 (HEK293) cell lines that stably express hERG1a (Kv11.1) were generated by using a pCEP4 plasmid that contained hERG cDNA and transfected by using a lipofectamine method (Thermo Fisher Scientific, Waltham, MA, USA). A hygromycin resistance gene was used for the selection of stable lines. Single colonies were picked and examined for hERG currents by using whole-cell patch-clamp recordings (see below). Positive clones were cultured in minimum essential medium (Thermo Fisher Scientific) that was supplemented with fetal bovine serum (10%), nonessential amino acids (1%), an antibiotic antimycotic mix (1%), glutamax (1%; Thermo Fisher Scientific), and hygromycin (100 µg/ml; Calbiochem, San Diego, CA, USA). C723S mutation was introduced using the QuikChange site-directed mutagenesis kit (Stratagene, Cambridge, United Kingdom) according to manufacturer instructions. All constructs were verified by DNA sequence analysis.

The tick strain Media Joya-INIFAP was used to carry out infestations on a bovine, applying 10,000 15-day-old larvae kept in the laboratory at 28 °C with constant humidity. At 21 days post-infestation, engorged or semi-engorged females were collected and washed 3X with distilled water to remove hair and skin from the host. Three washes are carried out by immersion in 10% benzalkonium chloride for 10 min at room temperature and rinsing 3X with sterile distilled water. A final wash was performed with sterile distilled water and an antibiotic-antimycotic mixture (100X, Thermo Fisher Scientific, Waltham, MA, USA) at a ratio of 1:100 for 10 min at room temperature and two rinses with sterile distilled water. Ticks were dried on a bed of sterile gauze. The embryonic cells from R. microplus strain Su-INIFAP [44 (link)], were cultured on a 25 cm² cell culture flask (Corning, Corning, NY, USA) in 5 mL of Leibovitz’s L-15 -MEM (1:1) (Thermo Fisher Scientific) supplemented with 20% fetal bovine serum (Gibco) and antibiotic-antimycotic mix (100X, Thermo Fisher Scientific) and incubated at 32 °C and 3% CO₂. The cells in the log growth phase were harvested and washed by centrifugation at 8000× g in PBS (Sigma, St. Louis, MO, USA) and quantified (Nabi-UV/Vis Nano Spectrophotometer, MicroDigital, Seoul, Republic of Korea) before RNA extraction.