Experimental Autoimmune Myocarditis:
Myocarditis was induced by immunization with cardiac α-myosin heavy chain (AnaSpec Inc., Fremont, CA, USA) as explained previously . 100 µg cardiac α-myosin heavy chain was dissolved in 100 µL sterile phosphate buffered saline (PBS) and mixed with 100 µL of complete Freund’s adjuvant (Sigma, F5881, st. Louis, MO, USA). This mixture was vortexed for 1.5 h to create an emulsion of antigen in complete Freund’s adjuvant. On day 0, 200 µL of emulsion was injected subcutaneously at two flanks of wildtype and PKP2-Hz mice. For this study, mice were backcrossed onto the BALB/c background as it is permissive for myocarditis . In addition, the mice were injected intraperitoneally with 500 ng of pertussis toxin (List Biological Laboratories, Campbell, CA, USA) dissolved in PBS. The mice were monitored for at least one week following the injections. A mouse was euthanized if it showed unusually large granulomas, ulcerations, or signs of being moribund. After three or six weeks, echocardiograms and electrocardiograms were recorded, the mice were sacrificed by CO2 euthanasia and the hearts were removed for histological studies.
Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides Product #180 - Pertussis Toxin from B. pertussis, Lyophilized in Buffer and Product #181 - Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free).
THP-1 differentiation and stimulation.:
THP-1 monocytes were added to 24-well tissue culture plates at 105 cells per well in 1 ml Roswell Park Memorial Institute medium supplemented with 20% fetal bovine serum. Suspension monocytes were converted to adherent macrophage-like cells by addition of 50 ng/ml phorbol myristate acetate (PMA; Sigma, St. Louis, MO, USA) and incubation at 37°C in 5% CO2 for 48 h. Differentiation of monocytes into macrophage-like cells was confirmed by visual inspection of the cellular morphology, confirmation of adherence, and increased cytoplasmic volume. The differentiated THP-1 cells were stimulated with the addition of 1.5 × 107 CFU/well of live or heat-inactivated bacteria added in isolation or in combination with 1 μg/ml purified CT or CTB (both from List Biologicals, Campbell, CA, USA). ...
Author did not specify which Cholera toxin was utilized. List Labs Product #101B/C - Cholera Toxin from Vibrio Cholerae has been discontinued; however, List Labs still provides Product #100B - Cholera Toxin (AZIDE-FREE) from Vibrio cholerae.
To follow, are our recommendations for transitioning from Product #101B/C to Product #100B:
Product #101B, Cholera Toxin from Vibrio cholerae (1 mg), has been discontinued. Product #100B, Cholera Toxin (AZIDE-FREE) from Vibrio cholerae, may be substituted for Product #101B, if the user accounts for the formulation differences. Both products contain the same quantity of toxin per vial (1.0 mg). For Product #100B, reconstitution with 1.0 mL of water results in a 1 mg/mL toxin solution in a buffer with the following component concentrations: 0.05 M Tris, 0.2 M NaCl, 0.001 M Na2EDTA at pH 7.5. For Product #101B, reconstitution with half that amount of water (0.5 mL) resulted in a 2 mg/mL toxin solution with the same concentrations of the buffer components, and in addition, Product #101B contained 0.003 M NaN3. The user may add the sodium azide preservative to product #100B, if desired.
Product #101C, Cholera Toxin from Vibrio cholerae (2 mg), has been discontinued. Product #100B, Cholera Toxin (AZIDE-FREE) from Vibrio cholerae, may be substituted for #101C, if the researcher accounts for the formulation differences. While Product #101C contained 2.0 mg per vial, Product #100 contains 1.0 mg per vial. When Product #100B is reconstituted with 1.0 mL of water, the resulting 1 mg/mL toxin solution will have buffer components with the following concentrations: 0.05 M Tris, 0.2 M NaCl, 0.001 M Na2EDTA at pH 7.5. For Product #101C, reconstitution with less than half that amount of water (0.4 mL) resulted in a 5 mg/mL toxin solution in the same buffer, and in addition, Product #101C contained 0.003 M NaN3. The user may add the sodium azide preservative to product #100B, if desired.
Large quantities of Product #101B or Product #101C may be obtained through Bulk Requests and Customer Orders.
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 μl 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.
... anti-CTB at 1:10,000 (List Biological Laboratories, Campbell, CA). ...
... CTb, Goat, 703, 1:1000, List Biological. ...