A549 cells and H1299 NSCLC cell lines were cultured in a humidified tissue incubator at 37°C under 5% CO2. A549 cells and H1299 cells were incubated with Kaighn’s Modification of Hams F-12 (F-12K; Corning, 10-025-CV) and Roswell Park Memorial Institute (RPMI; Corning, 10-043-CVR) media, respectively. Cells were treated with 250 pM TGFβ1, 10 μM ULK-101, 10 μM Compound C, 20 μM SB431542, 40 μM LY294002 and 10 μM P38 MAPK Inhibitor in media supplemented with 10% FBS. Transient siRNA knockdowns were performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778150) and optimem media (Thermo Fisher Scientific, 22600134) as per the manufacturer’s protocol. Stable GFP-LC3-RFP-LC3ΔG expressing cells were generated using PolyJet transfection reagent (Froggabio, Toronto, ON, Canada) and a cDNA pMRX-IP-GFP-LC3-RFP-LC3ΔG vector (Addgene, 84573). Transfected cells were isolated using growth media supplemented with 10% FBS and 1 μg/mL puromycin (Thermo Fisher Scientific, A1113802).
Polyjet transfection reagent
Manufactured by FroggaBio
Sourced in Canada
PolyJet is a transfection reagent used for the introduction of nucleic acids, such as plasmid DNA or siRNA, into a variety of mammalian cell lines. It facilitates the uptake of these molecules by cells, enabling their expression or silencing within the cellular environment.
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Lab products found in correlation
7 protocols using polyjet transfection reagent
1
Autophagy Regulation in NSCLC Cells
2
Production and Characterization of Pseudotyped HIV Particles
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PV was generated by co-transfecting plasmids encoding an Env-deficient HIV-1 backbone (pSG3ΔEnv, NIH ARP #11051), CD14 (pCD14, AddGene #13645), and/or the HIV-1 envelope glycoprotein (pBaL.01, NIH ARP #11445) into 293T cells. In brief, cells were plated at 3 × 105 cells/well in 12-well plates (1 mL complete DMEM) and allowed to adhere overnight. The next morning, plasmid DNA was diluted and mixed with Poly Jet transfection reagent following the manufacturer’s instructions (Frogga Bio #SL100688). For WT PVs, 0.5 µg SG3ΔEnv plasmid was transfected alone, and for CD14+ PVs, 0.5 µg SG3ΔEnv + 0.25 µg CD14 plasmids were co-transfected. Cells were cultured for 48 h after transfection, after which cell culture supernatants containing virus particles were collected, centrifuged to remove cellular debris, and frozen at −30°C until use.
3
Lentiviral Transduction and Transfection of 293T Cells
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For transfection, 7×105 293 T cells per well were cultured in 1 ml culture media in a 12-well plate for 1 day. Media were exchanged and 1 h later each well was transfected with 0.75 μg of pLV-CMV-AAT-sCD4 using PolyJet transfection reagent (FroggaBio, North York, Canada) according to the manufacturer’s instructions. For transduction, 1×105 293 T cells were mixed with the lentiviral vector at the indicated multiplicity of infection (MOI) and polybrene (final concentration 8 μg/ml) in a 12-well plate in a 1 ml volume. The cells were incubated for 3–4 days. The cells were sub-cultured for 1–2 weeks and then used in subsequent experiments.
4
FLNa Expression in 3T3 Fibroblasts
5
BiFC and Subcellular Localization in HeLa Cells
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For BiFC and subcellular localization studies, 2.5×105 HeLa cells were seeded onto coverslips and 24 hours later plasmids were transfected into cells at equal molar ratios using PolyJet transfection reagent (FroggaBio, Toronto, Canada). Twenty-four hours post transfection cells were incubated for one hour at room temperature to allow for fluorophore maturation, as described previously22 (link). Subsequently, cells were fixed in 4% PFA and prepared for immunofluorescence as described below.
6
Autophagy Regulation in NSCLC Cells
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H1299 cells and A549 NSCLC cells were purchased from ATCC and cultured in Roswell Park Memorial Institute (RPMI; Corning, 10-043-CVR) and Kaighn's Modification of Hams F-12 (F-12K; Corning, 10-025-CV) media, respectively. Both cell lines were passaged using 0.25% trypsin EDTA (Sigma-Aldrich, T2605), centrifuged at 1000×g for 2 min and resuspended in fresh media supplemented with 10% FBS. A humidified tissue incubator cultured the cells at 37°C under 5% CO2. Cells were treated with 250 pM TGFβ1, 50 µM chloroquine, 10 µM spautin-1, 10 µM MG132 in media supplemented with 10% FBS. Transient siRNA knockdowns in H1299 cells and A549 cells were performed using optimem media (Thermo Fisher Scientific, 22600134) and Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778150) as per the manufacturer’s protocol. Stable GFP-LC3-RFP-LC3ΔG expressing cells were generated using a cDNA pMRX-IP-GFP-LC3-RFP-LC3ΔG vector (Addgene, 84573) and PolyJet transfection reagent (Froggabio, Toronto, ON, Canada). Transfected cells were isolated using 1 µg/ml puromycin (Thermo Fisher Scientific, A1113802) in growth media supplemented with 10% FBS.
7
Quantifying Intracellular and Extracellular ACTH
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To quantify intracellular and extracellular levels of ACTH, 3 x 10 5 AtT-20 cells were seeded into 12 well dishes and 24 hours later, cells were transfected with 2.5μM siRNA using PepMute TM (FroggaBio, North York, Canada). Forty-eight hours post-transfection cells lysates and supernatants were collected for Western blot and MAGPIX analysis, respectively. For microscopy, 3 x 10 5 AtT-20 cells were seeded on coverslips and transfected with 2.5µM siRNA and 1µg of the appropriate plasmid DNA using PepMute TM siRNA or PolyJet Transfection Reagent (FroggaBio). Forty-eight hours post transfection, cells were fixed and prepared for imaging.
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