Lps eb ultrapure

LPS wea electroporated into podocytes using program T-020 in Nucleofector 2b (Lonza, Valais, Switzerland) and Amexa Basic Nucleofector Kit for Primary Mammalian epithelial cells (VPI-1005) according to manufacturer’s instruction. LPS-EB ultrapure (Invivogen, San Diego, CA, USA) was added to differentiated podocytes at a concentration of 3.0 ng/400,000 cells. The supernatants were collected at 4 h after electroporation and protein from them were collected using Amicon Ultra (Merck, Darmstadt, Germany) and analyzed with western blotting. Samples were normalized to mL/number of cells in the medium. Z-VAD-fmk (Selleck Biotech, Tokyo, Japan) was added 1 h prior to, during and after electroporation at a concentration of 20 nM.

PBMCs were seeded at 1×10⁶ cells/ml in a 24-well plate and stimulated with the following TLR ligands at the concentration of 1 µg/ml Pam3CSK4 (TLR2), 10 µg/ml polyIC (TLR3), 0.5 µg/ml LPS-EB ultrapure (TLR4), 1 µg/ml CLO75 (TLR7/8), 4 µg/ml CpG (ODN 2006) (TLR9) (all Invivogen, San Diego, CA, USA) or PBS (HyClone, Thermo Scientific) for 24 hours.
For inhibition of TLR3 signalling, PBMCs were pre-treated for 6 hours with 5 µM PepinhTRIF (Invivogen) and subsequently stimulated with polyIC as described above.

Stably transfected cell line HEK 293-TLR4/MD2-CD14 or HEK 293 hTLR2 were seeded into 96-well plates at the concentration of 3 × 105 cells/mL. The next day cells were transiently transfected with PolyFect Transfection Reagent (Qiagen, Hilden, Germany) with a reaction mix composed by 150 ng of Firefly luciferase reporter constructs, pGL3.ELAM.tk (containing NF-κB promoter sequences), and 15 ng of Renilla luciferase reporter plasmid, pRLTK (as an internal control). Twenty-four hours post-transfection cells were untreated or incubated with different concentrations (1, 10, and 100 ng/mL) of R-LPS of H. lacunaris TB21 or of purified E. coli LPS (LPS-EB ultrapure; InvivoGen, San Diego, California, USA) for 4 h to analyze the NF-κB activity or for 18 h to measure the IL-8 release, respectively. For the competition assays, HEK 293-TLR4/MD2-CD14 cells were primed with LPS (1, 10 and 100 ng/mL) of R-LPS of H. lacunaris TB21 for 1 h and then stimulated with 10 or 100 ng/mL of E. coli LPS for 4 h. After this time, the NF-κB activity and IL-8 release were measured.
HEK 293 hTLR2 were exposed to R-LPS of H. lacunaris TB21 as above and Pam3CSK4 (1 µg/mL, InvivoGen, San Diego, CA, USA) was used as the positive control. After 6 h, NF-κB activity was measured and IL-8 release was recorded via ELISA assay after 18 h of stimulation.

Resting microglia (BV-2) cells generally do not have high uptake of nanoparticles^{59 (link)} unless they are activated by a signal such as lipopolysaccharide (LPS)-stimulated inflammation.^{60 (link)} As a control for uptake, 5000 BV-2 microglia cells were sub-cultured in complete DMEM overnight onto sterile coverslips (22 × 22 mm²) in 6-well plates as described in Section 2.5. Cells were treated with LPS (500 ng mL⁻¹; LPS-EB Ultrapure; InvivoGen) and incubated for 4 hours; cells were then washed with 1% (v/v) PBS and 10 μg of raw–FNDs (1 mg mL⁻¹) in 2 mL of complete media and incubated at 37 °C and 5% CO₂ for 24 hours. Then, cells were washed with 1% (v/v) PBS, fixed and mounted on microscopy slides following the protocol described above.

Antibodies used include: GSDMD Rabbit polyclonal Ab (Novus Biologicals, NBP2–33422), FLAG® M2 monoclonal Ab (Sigma, F3165), GAPDH Rabbit monoclonal Ab (Cell Signaling Tech, 14C10), CASP1 p20 Rabbit polyclonal Ab (Cell Signaling Tech, 2225s), CASP1 p12/10 Rabbit monoclonal Ab (Abcam, ab179515), CASP4 Rabbit polyclonal Ab (Cell Signaling Tech, 4450S), CASP5 Rabbit monoclonal Ab (Cell Signaling Tech, 46680S), Myc Mouse monoclonal Ab (Cell Signaling Tech, 2276S), V5 Rabbit monoclonal Ab (Cell Signaling Tech, 13202S), hIL-1β Goat Polyclonal Ab (R&D systems, AF-201-NA), hIL-18 Goat Ab (R&D systems, af2548), hIL-1α Recombinant Ab (PeproTech, 200–01A), PARP Rabbit polyclonal Ab (Cell Signaling Tech, 9542S), HA Rabbit monoclonal Ab (Cell Signaling Tech, 3724S), mIL-1β Goat polyclonal Ab (R&D systems, AF-401-NA), CASP11 Rat monoclonal Ab (Novus Biologicals, NB120–10454), FKBP12 Rabbit polyclonal Ab (Abcam, ab24373). IRDye 800CW anti-rabbit (LICOR, 925–32211), IRDye 800CW anti-mouse (LI-COR, 925–32210), IRDye 680CW anti-rabbit (LI COR, 925–68073), IRDye 680CW anti-mouse (LI-COR, 925–68072). Other reagents used include: LPS-EB Ultrapure (Invivogen, tlrl-3pelps), VX-765 (Apexbio Technology LLC, 50–101-3604), Z-VAD-FMK (Enzo Life Sciences, NC9471015), FuGENE HD (Promega, E2311), AP20187 (Tocris™ 6297/5), NP-40 Lysis Buffer Low Salt (Thomas Scientific, C994H79).