Lipofectamine messengermax reagent

To prepare for Western blot, 293 T cells (ATCC) were seeded at 3 × 10⁵ cells/well in 24-well plates in Opti-MEM (Thermo Fisher Scientific 51985091), 10% fetal calf serum, 1% Antibiotic-Antimycotic containing penicillin (used at 100 units/mL), streptomycin (used at 100 μg/mL), Amphotericin B (used at 0.25 μg/mL) Gibco Cat. #15240-062). Cells were incubated overnight to ≈80% confluence. Cells were then transfected with two different mRNA quantities (500 ng or 1,500 ng) using Lipofectamine MessengerMax reagent (Thermo Fisher LMRNA001) following the manufacturer’s recommendations. At 48-h posttransfection, supernatants were removed and cell monolayers disrupted with 100 μL of 1× passive lysis buffer (Promega E194A). Insoluble debris was pelleted by centrifugation.
For ELISA, 293 T cells were seeded at 2 × 10⁴ cells/well in 96-well plates for overnight incubation, resulting in ~80% to 90% confluence. Transfection reagents and conditions were again as for the Lipofectamine MessengerMax reagent per manufacturer’s recommendations, but with 100 ng per well of RNA generated by GreenLight Biosciences or with commercially available in vitro transcription reactions. After overnight incubation, cell monolayers were used as targets in an ELISA.

We compared the translation efficacy of the GFP mRNAs with the UTRs HBA1, HBA2, and HBB in transfected cAT-MSCs. Cells (1 × 10⁵) were seeded in confocal dishes and transfected with 0.5 μg of each mRNA for 24 h with Lipofectamine MessengerMAX Reagent (Thermo Fisher Scientific) and Opti-MEM I Reduced Serum Medium (Gibco) according to the manufacturers' instructions. The fluorescence levels of transfected cells were measured using a confocal laser scanning microscope (LSM710; Zeiss, Germany) at 6, 12, 24, 36, 48, 60, 72, 84, and 96 h post-transfection. At each time point, three images per confocal dish were randomly acquired. The relative fluorescence was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Briefly, we evaluated the area and intensity of green fluorescence using the ImageJ as the cells were evenly seeded. The fluorescence area was relatively calculated by assuming the total area as 1.

High-molecular weight (HMW, #tlrl-pic) and low-molecular weight (LMW, #tirl-picw) poly(I:C) dsRNAs, produced by InvivoGen (San Diego, CA, USA), were used as PRR agonists. Total RNA was isolated from either uninfected A549 cells or cells infected with IAV A/California/07/09 (H1N1pdm09) (24 h post infection). Total RNA was extracted from cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in full accordance with manufacturer’s instructions. Viral genomic RNA was isolated from influenza virus A/California/07/09 (H1N1pdm09) using the RNeasy Mini Kit (Venlo, Netherlands). RNA transfections were performed using Lipofectamine MessengerMAX Reagent (Thermo Scientific, Waltham, MA, USA) according to manufacturer instructions. There were approximately 0.6 µL of Lipofectamine Messenger Max Reagent and 1.40 × 10¹¹ RNA molecules per well. For IVT RNA-GFP, total RNAs, mRNA, viral genomic RNA, LMW, and HMW, this was approximately 80, 160, 16, 40, 120, and 380 ng, respectively. The immunogenicity of the studied RNAs was evaluated 24 h after introduction of the complexes.

Three 27 nucleotide siRNA duplexes specific for ALOXE3 (Table 1) and a TYE-563 tagged transfection control were purchased (DsiRNA TriFECTa Kit; Integrated DNA Technologies, Coralville, IA, USA) and reconstituted according to the manufacturer’s protocol. Transfection of the DsiRNAs into EA.hy926 cells was achieved using Lipofectamine MessengerMAX Reagent (ThermoFisher Scientific, Waltham, MA, USA). The transfected cells were incubated for 48 h, at which time brightfield and fluorescent images were captured with an Axiovision 4.1 Zeiss microscope. Cells were subsequently harvested, and ALOXE3 was measured.

The transfection was conducted according to the manufacture’s protocol. Jurkat cells (1 × 10⁵ cells/mL, 2 mL) were seeded in 3.5 cm dishes. Lipofectamine MessengerMAX reagent (ThermoFisher Scientific, Waltham, MA, USA) and the mRNA were mixed in the Opti-MEM (ThermoFisher Scientific, Waltham, MA, USA) at a ratio of 1.5 µL reagent/µg mRNA (0.5 µg). The mixture was incubated for 5 min. After the incubation, the mixture was transfected to the Jurkat cells in culture media containing serum.

To prepare for Western blot, 293 T cells (ATCC) were seeded at 3 × 10⁵ cells/well in 24-well plates in Opti-MEM (Thermo Fisher Scientific 51985091), 10% fetal calf serum, 1% Antibiotic-Antimycotic containing penicillin (used at 100 units/mL), streptomycin (used at 100 μg/mL), Amphotericin B (used at 0.25 μg/mL) Gibco Cat. #15240-062). Cells were incubated overnight to ≈80% confluence. Cells were then transfected with two different mRNA quantities (500 ng or 1,500 ng) using Lipofectamine MessengerMax reagent (Thermo Fisher LMRNA001) following the manufacturer’s recommendations. At 48-h posttransfection, supernatants were removed and cell monolayers disrupted with 100 μL of 1× passive lysis buffer (Promega E194A). Insoluble debris was pelleted by centrifugation.
For ELISA, 293 T cells were seeded at 2 × 10⁴ cells/well in 96-well plates for overnight incubation, resulting in ~80% to 90% confluence. Transfection reagents and conditions were again as for the Lipofectamine MessengerMax reagent per manufacturer’s recommendations, but with 100 ng per well of RNA generated by GreenLight Biosciences or with commercially available in vitro transcription reactions. After overnight incubation, cell monolayers were used as targets in an ELISA.

cDNA for in-vitro generated mRNA constructs was obtained from expansion of an ATF4 Mammalian Gene Collection Escherichia Coli strain (Horizon Gene Editing, Clone ID: 3454473), while G. Extracted DNA was PCR amplified using primers listed in Table S4 to generate ATF4 WT or Mut constructs, purified (PCR Cleanup Kit, Qiagen) and mRNA was generated using mMESSAGe mMACHINE T7 Ultra Kit (Ambion) according to manufacturer’s instructions. 500 ng of generated mRNA was transfected into Cyc or Etop cells using 4 μl Lipofectamine MessengerMAX Reagent (ThermoFisher Scientific). Cells were incubated for 12 hours until harvest for bulk RNA and protein extraction.