A purified complex of metabolically labeled endotoxin ([3H] lipooligosaccharide LOS) and

A purified complex of metabolically labeled endotoxin ([3H] lipooligosaccharide LOS) and insect-cell derived recombinant human being myeloid differentiation element 2 (MD-2) [3H] LOS·MD-2 has been used to demonstrate pM affinity binding interactions with soluble Toll-like receptor 4 ectodomain (TLR4ecd). of [125I] to sMD-2 with 20-collapse higher specific radioactivity and TLR4-activating properties comparable to metabolically labeled LOS·MD-2. LOS·MD-2[125I] and [3H]LOS·MD-2 have related affinities for soluble FLAGTLR4ecd and for membrane-bound TLR4 in transiently transfected EPI-001 HEK293T/TLR4 cells. In a similar dose-dependent manner sMD-2 and LOS·MD-2 inhibit LOS·MD-2[125I] binding to TLR4 indicating the pM affinity binding of LOS·MD-2[125I] is definitely agonist-independent. LOS·MD-2[125I] allowed measurement of low levels of cell-surface human being or murine TLR4 indicated by stable cell lines (2 0 0 sites/cell) and quantitatively steps low levels of “MD-2-free” TLR4 (est. 250 molecules/cell) in cells co-expressing TLR4 and MD-2. Profession of 50-100 TLR4/cell by LOS·MD-2 is sufficient to result in measurable TLR4-dependent cell activation. LOS·MD-2[125I] provides a powerful reagent to EPI-001 measure quantitatively practical cell-surface TLR4 in EPI-001 human being and murine cells including cells where surface TLR4 are potentially functionally significant but too low to detect by additional methods. serogroup B (NMB) produced in Morse medium supplemented with either 2 mM 1-[12C] 2 (Moravek Chemicals Brea CA) and 1 uCi/ml of 1 1 2 acetate or 2 mM sodium acetate comprising 5 mCi/ml [3H]acetate (Moravek Chemicals Brea CA) as previously explained.22 23 Human being serum albumin (HSA) was an endotoxin-free 25 stock answer from Baxter Health Care (Glendale CA). Chromatography matrices (Sephadex G10 Sephacryl HR S100 and 200 and Ni2+ FF-Sepharose) were purchased from GE Healthcare Piscataway NJ). Bovine serum albumin EPI-001 anti-FLAG M2 Rabbit polyclonal to SORL1. agarose and additional chemical reagents were from Sigma. Iodogen was purchased from Pierce (Rockford IL) and used according to the manufacturer’s instructions. The stable HEK293 cell collection expressing EPI-001 human being TLR4 was a nice gift from Dr. Jesse Chow (Eisai Study Institute Andover MA) and the stable HEK293 cell collection expressing murine TLR4 was purchased from Invivogen (San Diego CA). Conditioned medium comprising secreted FLAG-tagged TLR4 ectodomain-vlr-Fc protein used in capture assays was from Freestyle HEK293F cells stably transfected with TLR4 (amino acids 27-527) inside a CMV vector comprising additional hagfish amino acid sequence in the C-terminal end followed by the Fc website. The stable transfected cells were a generous gift of Dr. Richard Tapping University or college of Illinois Urbana IL. Control conditioned medium (i.e. not comprising FLAGTLR4ecd) from HEK293 cells was used as a negative control. The pCMV-FLAG-TLR4 vectors encoding either TLR4 wt or mutant D299G.T399I were a generous gift of Dr. Stefanie Vogel Univ. of Maryland Baltimore MD. The IL-8 ELISA kit (BD OptEIA) was purchased from BD Biosciences (San Diego CA) and used relating to manufacturer’s instructions. Binding data were analyzed using GraphPad Prism 5 Software (La Jolla CA). Preparation of recombinant human being MD-2 Recombinant human being His6-MD-2 was generated as previously explained9 except that cDNA encoding human being MD-2 was optimized for transcription in Large Five cells (and restriction sites into the baculovirus transfection vector pBAC3 (Novagen Billerica MA) that contains a six-residue polyhistidine (His6) tag in the N-terminal end and a 5′ flanking transmission sequence (gp64) to promote secretion of the indicated protein from your insect cells. Baculovirus stocks comprising the gene for His6-MD-2 were generated by transfection of BacVec3000 and plasmid into Sf9 cells using the BacVec3000 kit from Novagen according to the manufacturer’s training. The generated baculovirus was then amplified in Sf9 cells and used to infect Large Five? (Invitrogen Grand Island NY) insect cells in serum-free medium for protein production. Large level (20 L) preparations of conditioned insect medium comprising secreted His6-MD-2 were produced by BlueSky Biotech Worcester MA. Preparation of LOS·MD-2 complexes We have recently explained the preparation of endotoxin·albumin complexes (Mr ~ 70 0 and conditions in which endotoxin·albumin complexes react with conditioned insect medium comprising sMD-2 to generate endotoxin·MD-2.18 24 [14/13C]LOS·albumin complexes (15 cpm/pmol) were generated from [14/13C]LOS aggregates dispersed in 100 mM.