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Lethal Factor (20 nM final concentration) and MAPKK substrate (MAPKKide 12.5 M, final concentration) were purchased from List Biological Laboratories, Campbell, CA and used according to the fluorescence resonance energy transfer (FRET) method. ...
Recombinant PA83 and PA63 were purchased from List Laboratories (New Jersey). PA63is a cleavage product that is capable of binding LF (45). For the sandwich ELISA, 50 l of PA at 63 kDa and 83 kDa (6 g/ml) was applied to a 96-well plate and blocked with 2% milk-PBS as described above. For initial assays, LF was diluted in PBS or human serum at 5 g/ml. For assays detecting LF in infected animals, serum was added to the plate initially diluted 1:1 in 2% milk-PBS and then serially diluted across the plate in duplicate. After a 1-h incubation, the plate was washed as described above. Goat anti-LF polyclonal serum (List Labs) was diluted 1:1,000 in 2% milk-PBS and added to the plate for a 1-h incubation in duplicate. The plate was then washed, followed by the addition of goat anti-rabbit IgG-HRP conjugate (Bio-Rad) diluted in 2% milk-PBS for 1 h. ELISA reactions were developed with OPD tablets (Sigma) and quenched by the addition of 50 l of 4.5 M H2SO4. ...
Recombinant anthrax PA and LF were purchased commercially and were stored in 1:1 glycerol-water at 20C (List Biological Laboratories) for in vitro studies. Unless otherwise indicated, anthrax LT was administered in excess at concentrations of 2.5 g/ml PA and 1 g/ml LF. In selected experiments a proteolytically inactive mutant of LF was used as a negative control (E687C substitution in zinc binding site that eliminates enzymatic activity; List Biological Laboratories). ...
Anthrax PA binding assays:
Purified murine or human B cells were cultured at 4C for 30 min with FITC-labeled anthrax PA (50 g/ml; List Biological Laboratories) in the presence or absence of unlabeled anthrax PA (150 g/ml) to confirm specific binding. Stained cells were then washed with PBS and analyzed by flow cytometry (see below). Unstained cells were analyzed in parallel to establish background levels of autofluorescence.
Primary B cells were cultured in complete RPMI for 4 to 5 h, washed with RPMI 1640, and then stimulated as indicated in the presence or the absence of anthrax LT for 7 days. ...
Murine in vivo studies:
Mice were treated with varying doses of anthrax LT as indicated, using a fixed ratio of LF/PA of 1:2.5. LF and PA were resuspended in PBS and injected i.p. into mice in a total volume of 1.0 ml of PBS. As a negative control, selected mice were treated with PBS alone. Mice were sacrificed 3 h after treatment, and spleens were harvested for primary B cell isolation as previously described. Primary B cells were then evaluated for proliferation and IgM production.
Mice were immunized with an acapsular mutant of S. aureus Reynolds designated JLO22 (7) that was cultivated in TSB to the logarithmic phase of growth (A650, 0.34). The bacteria were pelleted, suspended in phosphate-buffered saline at a concentration of 108CFU/ml in an open petri dish, and exposed to a UV light source that was 8 cm away for 10 min on a rotator in the dark. The bacteria were concentrated by centrifugation, and 10 l of the UV-killed bacterial suspension containing 108 CFU S. aureus with or without 5 g of cholera toxin B (CTB) (List Biological Laboratories, Inc., Campbell, Calif.) was applied to each mouse nose on days 0, 5, and 10. CTB was omitted from the third immunization to reduce nonspecific protection. Two weeks after the third immunization, the mice were inoculated with 108 CFU S. aureus strain Newman cultivated in TSB to the logarithmic phase (A650, 0.34). Colonization was evaluated after 14 days.
Author did not specify which CTB was utilized. List Labs has provided Product #103B (Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae) and Product #104 (Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt). The former has been discontinued. The latter may be substituted for the former, if the researcher takes into account the difference in the buffer in which each product is provided and the amount of protein per vial. When Product #103B was reconstituted with 200 l of water, it contained 1 mg of protein in 0.05 M Tris, 0.2 M NaCl, 0.001 M Na2EDTA, 0.003 M NaN3, pH 7.5. When Product #104 is reconstituted with 0.25 ml water, it contains 0.5 mg of Cholera Toxin B Subunit (choleragenoid) in 0.01 M Sodium Phosphate at pH 7.5.
Granulocyte-enriched populations of bovine peripheral leukocytes were purified as described above and suspended to 1.2 107 cells/ml in HBSS (137 mM NaCl, 5.6 mM glucose, 5 mM KCl, 4 mM NaHCO3 1 mM CaCl2, 0.5 mM MgCl2, 0.4 mM KH2PO4, 0.4 mM Na2HPO4, and 0.4 mM MgSO4 (pH 7.4)). Aliquots of the cell suspension were incubated for 60 min at 37C in a final volume of 500 l containing one of the following stimulants: 100 nM PMA, 20 g/ml lipoteichoic acid (LTA) from Bacillus subtilis, 160 g/ml muramyl dipeptide (MDP) (Sigma-Aldrich), 100 g/ml LPS from Staphylococcus typhimurium (List Biological Laboratories), ...
... Flat bottom microtiter plates (Nunc F96 Maxisorp) were coated with 50 l of Bacillus anthracis Protective Antigen (PA) or Lethal Factor (LF)(List Biological Laboratories (Campbell, Calif.) at a concentration of 1 g/mL in PBS overnight at 4 C. ...
... Two of the 800 cGy-irradiated mice (mice #26 and #27) and four naive E16 mice were immunized with tetanus toxoid (List Biological Laboratories, Inc., Campbell, CA, USA) 3 weeks after the final FVIII injection as follows: 4 g tetanus toxoid was mixed with 1 mg Al (OH)3adjuvant (Alhydrogel 2%; Accurate Chemical & Scientific Corporation, Westbury, NY, USA), diluted in sterile saline to a total volume of 108 l and injected intraperitoneally. Two weeks later, sera from these mice was assessed for the presence of anti-tetanus toxoid antibodies by ELISA (described below).
... Recombinant anthrax LF was obtained from List Biological Laboratories (Campbell, California, United States). PA was generously provided by Dr. Steven Leppla (National Institutes of Health). PA and LF were reconstituted in water at 500 ng/ml and 250 ng/ml, respectively . Recombinant LF and PA were produced in B. anthracis and were free of LPS contamination as indicated by the manufacturer, and by a lack of CD86 up-regulation on immature cells, as measured by flow cytometry.
Cell death and viability assays:
Cell viability was measured by analysis of MTT cleavage to formazan by succinate dehydrogenases in living cells . For the colorimetric MTT assay, cells were exposed to LT (500 ng/ml PA and 250 ng/ml LF), and the MTT solution (5 mg/ml MTT in PBS) was added directly to wells and incubated at 37 C for 4 h. The dye was solubilized with acidic isopropanol (25 mM HCl and 0.5% SDS in isopropanol), and the absorbance was measured at 570 nm. ...
Cells were cultured in 24-well plates and treated with LT. ...
In vivo assay:
Ten-week-old female BALB/c mice were injected intraperitoneally with LT [3,4]. Mice were sacrificed 0, 3, 6, and 24 h after LT injection. Spleens were harvested and treated with 400 U/ml collagenase D (Roche) to release DCs. Levels of splenic DCs (CD11c+, MHC class II+), macrophages (CD11b+, MHC class II+), and circulating B cells (B220+) and T cells (CD3+) were determined by flow cytometry using fluorescently labeled antibodies to surface markers.