Jetoptimus dna transfection reagent

HEK 293 T cells were transfected with the IFN-β luciferase reporter (IFN-β-Luc), TK-renilla, and HA-PARP9 plasmid or HA-vector by jetOPTIMUS DNA transfection reagent (Polyplus transfection) as per the manufacturer’s instructions. At 20 h after transfection, cells were left stimulated or stimulated for 10 h with LPIC, or viral dsRNA including Reo1187, Reo1198, Reo1320, and Reo1410 (1 μg/ml) delivered by Lipofectamine 3000 transfection (ThermoFisher Scientific), reovirus, or influenza virus infection. After stimulation or reovirus infection, cells were lysed and then measured using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions.

The plasmid for the mammalian expression of cytoplasm targeted ultrasensitive hydrogen peroxide indicator HyPer7 for optical imaging (pCS2+HyPer7-NES) was a generous gift from Vsevolod Belousov (IBCh, Moscow, Russia) (Addgene plasmid # 136467; http://n2t.net/addgene:136467 (accessed on 9 June 2022; RRID: Addgene_136467) [40 (link)]. Mesothelial and MPM cells were seeded into 2 mL plastic dishes, and HyPer7-NES transfection (3 μg DNA / dish) was performed when cells reached the 60–70% confluency using the JetOPTIMUS DNA transfection reagent (# 117-15, Polyplus transfection, Illkirch-Graffenstaden, France) according to the manufacturer’s instructions. Briefly, the medium was removed and substituted with Opti-MEM containing the plasmid DNA and the transfection reagent. Plasmid DNA (3 μg) was diluted in JetOPTIMUS Buffer (# 717-60, Polyplus transfection, Illkirch-Graffenstaden, France) and then mixed with JetOPTIMUS reagent according to the suggested ratio 1:1 between μg of DNA and μL of transfection reagent. After 10 min incubation at room temperature, the solution containing the DNA was added drop-wise to the cells and incubated at 37 °C for 4 h. Then, the media were exchanged for fresh culture media. All the experiments were performed 24 h after transfection.

HOS cells were co-transfected for 48 hours using jetOPTIMUS™ DNA transfection reagent (Polyplus-Transfection, Strasbourg, France) with 20 nM miRNA mimic or miRNA hairpin inhibitor and with BCL2-3’UTR vector (217HmiT016211a; 2761 bp), BCL2L1-3’UTR vector (217HmiT108616-MT05; 1489 bp) or MCL1-3’UTR vector (217HmiT127389-MT05; 2839 bp) (1 ng/µL; GeneCopoeia, Rockville, MD, USA) as previously described (27 (link)). These vectors contain the miR-342-5p and miR-4270 3’UTR sequences downstream a Gaussia luciferase (GLUC) reporter gene and a secreted alkaline phosphatase (SEAP) reporter gene. SEAP and GLUC luciferase activities were assayed in triplicate with the secrete-pair dual luminescence assay kit (GeneCopoeia, Rockville, MD, USA). Reporter activities were measured with a luminescence microplate reader (Spark 10M, Tecan) and expressed as relative luciferase units (RLU) corresponding to the GLUC : SEAP ratio.

HeLa cells were obtained from ATCC, cultured in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin (all PAN-Biotech GmbH, Aidenbach, Germany) and grown at 37 °C in a humidified incubator with 5% CO₂. Cells were transiently transfected with fluorescent-fusion constructs using jetOPTIMUS DNA transfection reagent (Polyplus transfection, Illkirch, France), according to the manufacturer’s protocol. A total of 24–48 h after transfection, cells were used for subcellular micropatterning experiments.

All
cell culture reagents
were purchased from Biochrom GmbH (Berlin, Germany). HeLa cells (ATCC)
were cultured in a RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin
and grown at 37 °C in a humidified incubator with 5% CO₂. For transient transfection, cells were sub-cultured the day before
and were then transfected with plasmids using the jetOPTIMUS DNA transfection
reagent (Polyplus transfection, Illkirch, France), according to the
manufacturer’s instructions.

The mouse CD47 or SIRPα promoters were cloned into a promoterless pGL4.20[luc2/puro] vector using Genentech’s cloning services (San Francisco, CA, USA). Vectors were transfected into SM1 and B16 melanoma cells using Lipofectamine 3000 (L3000008, ThermoFisher) and transfected cells were selected using puromycin. A31A7 macrophages were transiently transfected using jetOPTIMUS DNA transfection reagent (Polyplus, NY, USA). Luminescence was measured using the Luciferase Assay System (E4550, Promega) in a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices).