There're some papers talking about inducing non-canonical pyroptosis using DOTAP and LPS. However, the detailed are not provided. For example, according to one paper, 1 μg of LPS-SM and 15 μl of DOTAP were mixed together. What concentration should the LPS and DOTAP be when they're mixed together?
Does anyone know and be willing to provide the detailed protocol of LPS transfection or non-canonical pyroptosis induction?
Thanks!
From the supplementary material of this paper: http://science.sciencemag.org/content/sci/341/6151/1250.full.pdf
Liposome preparation and transfections
Bacterial lysate transfection complexes were generated as follows: For transfection of 5x104 BMMs, 0.25μL lysate suspended in 25μL OptiMEM and 0.25μL Lipofectamine 2000 suspended in 25μL were allowed to equilibrate at room temperature for 5 minutes. Suspensions were mixed and incubated at room temperature for 20 minutes. For LPS and lipid A transfection complexes, 75ng of LPS or lipid A suspended in 2μL OptiMEM and 375ng DOTAP suspended in 2μL OptiMEM were allowed to equilibrate at room temperature for 5 minutes. Suspensions were then mixed and incubated for 30 minutes at room temperature. Reaction volumes were then brought up to 50μL with OptiMEM.
Macrophage culture, treatment, and analysis of inflammasome activation
BMMs were prepared as described (26). For experiments, macrophages were seeded into 96-well tissue culture treated plates at a density of 5x104 cells/well. When indicated, macrophages were primed with lipopolysaccharide (50 ng/ml), poly(I:C) (1μg/mL) or IFN-γ (8 ng/ml ) overnight. For infections, culture media was replaced with OptiMEM and then bacteria were added to BMMs at MOI 200 (F. novicida) or MOI 5 (L. monocytogenes), centrifuged for 5 min at 200 x g, and incubated at 37°C. After 1 hour, extracellular bacterial growth was stopped by addition of 20 μg/ml gentamicin. In some experiments, 1μg/mL LPS or lipid A was included during the infection. For transfections, culture media was replaced with OptiMEM and then LPS and lysate transfection complexes were added to BMMs and centrifuged for 5 min at 200 x g. For CTB experiments, macrophages were primed for 5h with poly(I:C) and Pam3CSK4 (100μg/mL). Media was replaced with OptiMEM containing poly(I:C), Pam3CSK4, and combinations of CTB, LPS, and PrgJ as indicated in the figure legends. Supernatant samples were collected at the indicated time points. Cytotoxicity was defined as the percentage of total lactate dehydrogenase released into the supernatant and was determined as described (27). IL-1β secretion was determined by enzyme-linked immunosorbent assay (ELISA) (R&D Systems). PrgJ was purified as described (11). S. minnesota LPS was obtained from List Biologicals. E. coli O111:B4 LPS was from Sigma.
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