Glomax system

SMS libraries were prepared using the Nextera XT DNA Library Preparation Kit (# FC-131-1096, Illumina), following Illumina’s instructions (protocol # 15031942 v03 February 2018). Briefly, 1 ng of genomic DNA was used for the tagmentation reaction for a total volume of 20 µl. After 5 min at 55 °C, the reaction was stopped by adding 5 µl of the Neutralize Tagment (NT) Buffer. A limited-cycle PCR amplification was then performed to amplify the tagmentated DNA [addition of 15 μl of Nextera PCR Master Mix (NPM)] and to add Illumina sequencing adapters (addition of 5 µl of both Index 1 primer and Index 2 primer from the Nextera XT index kit, Illumina) for a total volume of 50 µl. The following PCR cycle program was used: 72 °C for 3 min, 95 °C for 30 s, 12 cycles of (95 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s), 72 °C for 5 min. SMS libraries were quantified using the QuantiFluor One dsDNA kit (# E4870, Promega) with the GloMax system (Promega). The quality of libraries was assessed using the High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer. Sequencing was performed on a NextSeq500 system (Illumina) with the NextSeq 500/550 High Output v2 kit (300 cycles) in 2 × 150 bp.

Drosophila Kc167 cells were cultured in Schneider's Drosophila Medium (Gibco) supplemented with 10% Fetal Bovine Serum (Gemini Bioscience). 250 ul of cells were seeded in 48 well plates, at a density of 1million cells/ml, and transient transfections were performed using Fugene transfection agent (Roche). Each well received 20 ng luciferase reporter vector and 2 ng pArmLacZ. Wnt signaling was activated by transfection with 10 ng pAcArm*, (a constitutively active Arm protein), and pAC5.1 EV was used as filler DNA to 100 ng total for each well. Cells were lysed and treated three days later using the Tropix Luc-screen kit (Applied Biosciences) and Luciferase and LacZ activity assayed using the Promega Glomax system. pArmLacZ was used to normalize for transfection efficiency.

To compare Wnt3a responsiveness in wild-type, Rab8a^−/− and Rab8b knockdown MEFs, cells were co-transfected with Topflash and Renilla luciferase for 24 h. The cells were then serum starved for 3 h in Wnt-free DMEM and treated with 20 ng/ml recombinant murine Wnt3a (Peprotech, 315-20). After 5 h medium was removed, cells washed with 1× PBS, lysed and luciferase activity detected using the dual-luciferase assay and Glomax system (Promega). To compare Wnt5a secretory abilities by wild-type and Rab8a^−/− MEFs, cells were simultaneously transfected with pcDNA-WNT5A, Topflash and Renilla luciferase in Wnt-free lactalbumin hydrolysate (SAFC Biosciences, 58901-C) in DMEM for 16-18 h. Topflash activity was detected in cell lysates and normalized to intracellular Renilla luciferase. Data represent three independent experiments with comparable transfection efficiencies.

Transient transfections of HEK-293/TLR-3, HEK293 and RAW264.7 cells were performed using GeneJuice (Merck-Millipore) according to manufacturer’s instructions. Stimulations involving poly I:C were performed for 6 hrs prior to lysis of cells. Cells were lysed in Passive Lysis Buffer (10mM EDTA, 100mM DTT, 50% glycerol, 5% Triton X-100, 125mM Tris base, pH 7.8). Luminescent activity was then measured on Promega GloMax system (Madison, WI, USA).

To determine intracellular ATP, sperm cells from the spermathecae of C. osakensis queens were introduced to 40 µL of 39.44 mM sodium sulfite in PBS (anoxic condition) and PBS (aerobic condition) for 1 min. Thereafter, the sperm samples were centrifuged at 5000 rpm for 5 min, after which the supernatants were removed, and CellTiter-Glo® Luminescent Cell Viability Assay buffer (Promega) was added. The sperm suspension was then divided into two samples: one for measuring relative luminescence unit (RLU) to determine ATP levels using a CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) and the other for measuring relative fluorescence unit (RFU) to assess DNA content using CellTox™ Green Cytotoxicity Assay kit (Promega). After the samples were prepared according to the manufacturer’s protocol, their luminescence and fluorescence were detected using the GloMax® system (Promega). We confirmed that the values were neither too low to detect nor saturated by checking whether they were within the linear range of the standard curve. To calibrate the ATP content by the number of sperm cells, we calculated values of RLU/RFU.

Cell viability was detected by CCK-8 kit (MedChemExpress, Shanghai, China). Cells (3,000/well) were cultured in 96-well plates for 48 h. After incubated with 10 μl CCK8 for 3–4 h, the absorbance was measured at 450 nm using GloMax^® System (Promega, WI, United States) (Chen et al., 2020 (link)).