Fetal bovine serum for culture of rat bone marrow macrophages was

Fetal bovine serum for culture of rat bone marrow macrophages was obtained from HyClone Lab oratories. all other tissue culture reagents have been from GIBCO BRL. Kinase assay kits, U0126, and antibodies against phosphorylated and non phos phorylated ERK1 and ERK2 had been obtained from Cell Sig nalling Technologies. All other reagents have been purchased from Sigma Chemical. Cells and bacteria Rat bone marrow derived macrophages have been isolated from female Sprague Dawley rats as previously described. Briefly, femurs had been removed from rats as well as the marrow flushed into 50 ml conical tubes. The cells were resuspended in DMEM and cultured in DMEM with 10% fetal bovine serum, antibiotics, and 10% L cell conditioned medium for 5?7 days. Macrophages were then removed from your culture dishes with cold EDTA and plated in 24 or 6 wells dishes as described for each experiment.
Before infection with BCG, the selleck chemicals media was altered to serum and antibiotic free of charge DMEM. For NFB experiments, bone marrow macrophages were pre pared from femurs of transgenic mice expressing a luci ferase gene driven through the HIV 1 prolonged terminal repeat containing 6 B consensus internet sites in its promoter. BCG, Pasteur strain, was obtained from the American Variety Culture Collection. Bacteria were cultured in Middlebrook Broth supplemented with OADC enrichment, and one. five ml aliquots of bacteria at around 108 bacteria per ml had been stored at 70 C. Colony forming units per ml were determined by plating serial dilutions with the bacteria onto Middle brook agar plates, and counting colonies just after 2?three weeks of growth. Purification of SP A SP A was purified from human alveolar proteinosis fluid or Dr. Samuel Hawgood as previously described. Briefly, one?2 ml of APF in PBS was extracted with 25 ml of 1 butanol after which dried overnight below nitrogen.
Dried protein was resuspended in 1 mM HEPES buffer, pH 7.five, with 0. 15 M NaCl and twenty mM n octyl D glucoside. The pel allow was collected by centrifugation at 17,000 g plus the practice repeated. The ultimate pellet was resuspended in five mM HEPES buffer with 1 mM EDTA and dia lyzed for 48 hours with buffer adjustments. 17AAG Just after dialysis, pol ymyxin B agarose was extra to the SP A along with the mixture was rotated for one hour at area temperature. The poly myxin B agarose was removed by centrifugation plus the SP A concentration was established utilizing the BCA pro tein kit from Pierce. The final SP A planning was divided into one ml aliquots and stored at four C for immedi ate use or twenty C for long lasting storage. The SP A was ana lyzed for purity by SDS Web page and for endotoxin contamination making use of the Limulus amebocyte lysate assay. Endotoxin ranges were rou tinely established to become lower than 0. 05 units/ml. Infections Frozen stocks of BCG had been thawed and vortexed vigor ously that has a glass bead to break up any clumps.

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