Transit jurkat transfection reagent

Zebrafish ZE cells were cultured at a density of 1 × 10⁶ cells in 60-mm dishes in 4 ml of Leibovitz's L-15 medium supplemented with 2% FBS. At 90% confluence, cells were transiently transfected with three different concentrations (1, 2, or 3 μg) of nSMase1 constructs (mock, wild-type, S270A mutant, and S270E mutant) or vector DNA using FuGENE 6 transfection reagent (Roche) following the manufacturer's instructions.
Jurkat T cells were cultured at a density of 2 × 10⁶ cells in 60-mm dishes in 4 ml of RPMI-1640 medium supplemented with 2% FBS. The cells were transfected with the nSMase1 mock, wild-type, S270A mutant, and S270E mutant constructs using TransIT Jurkat Transfection Reagent (Mirus Bio LLC, Madison, WI, USA). Briefly, 1, 2.5, or 5 μg of vector DNA was added to 10 μl TransIT Jurkat Reagent in 200 μl of serum-free RPMI-1640 medium, added to cells, and then incubated for 24 h at 37 °C. nSMase assays, the measurement of ceramide content, caspase-3 assays, and apoptotic DAPI analysis were then performed. Cell viability was determined using the Trypan Blue dye exclusion method.^{16 (link)}

Murine TLR2 promoter region (2000bp) was amplified by polymerase chain reaction (PCR) and cloned into the Firefly luciferase reporter plasmid pGL3-Basic (Promega) using EcoR I and Hind III restriction sites. RAW 264.7 cells seeded on 48-well plates were incubated until 60% to 70% confluence and then were transient transfected with 0.4 μg pGL3-TLR2 plasmid using TransIT – Jurkat Transfection Reagent (Mirus Bio). After transfection overnight, cells were left untreated or treated with DCA (100 μM) for 24 h, and methoctramine (5 μM) or SR 11302 (100 μM) was added 30 min ahead of DCA treatment if necessary. Cell lysates were collected for Luciferase activity determination by Dual Luciferase Reporter assay systems (Promega). According to the manufacturer’s instructions, luciferase activity was normalized by Renilla activity to standardize transfection efficiency. For the deletion analysis, a series of deletion mutants of the TLR2 promoter reporter plasmids (−1500, −1025, and −524/-1 bp) were constructed and applied.

Cells were grown to confluence in six-well plates and transfected with 1 μg of pBV-WT or pBV-MUT (Addgene) and 1 μg of the control pRL-SV40 vector (Promega) using TransIT^®-Jurkat Transfection Reagent (MIRUS) according to the manufacturer’s recommendations. Twenty-four hours posttransfection, the cells were lysed and assayed using the dual luciferase reporter assay system (Promega) on a Glomax 96 instrument (Promega).

Jurkat cells were transiently transfected with a TRPP2-siRNA plasmid, expressing specific PKD2 silencing sequences [12 (link)], by using the TransIT®-Jurkat Transfection Reagent (Mirus Bio, Tema Ricerca, Bologna, Italy). Briefly, cells were resuspended at 5 × 10⁵ cell density in 500 μL of complete medium and stratified on a 100 μL serum-free medium containing 1 μg of plasmid and 3 μL of transfection reagent. After 5 h of incubation, cells were pelleted by centrifugation at 800 g for 5 min and resuspended in fresh complete medium. As control, Jurkat wild type cells and transfected with scramble sequences were used.