Lethal toxin and signaling inhibitors:
Recombinant protective antigen and wild-type lethal factor or lethal factor mutant (E687C) were obtained from List Biologicals (Campbell, CA) …
In vitro lethal toxin assay with Rag1-/- splenocytes or human tonsillar lymphocytes:
100,000500,000 splenocytes per well were incubated in a round bottom 96 well plate in IMDM media supplemented with 10% FBS, 100 U/ml pencillin, 100 U/ml streptomycin, IL-2 (20 ng/ml) and IL-7 (10 ng/ml). Human tonsillar lymphocytes (2×106) were plated in 200 l RPMI media supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, non-essential amino acids, sodium pyruvate, IL-2 (20 ng/ml), IL-7 (10 ng/ml) and IL-1 (20 ng/ml). For initial lethal toxin experiments splenocytes/tonsillar lymphocytes were cultured in media containing in 0.1% serum to minimize the effect of serum on lethal factor enzyme activity. After the initial period of lethal toxin treatment (3 hrs), serum was replenished to a full concentration of 10%. Cells after toxin treatment were stimulated with recombinant mouse or human IL-23 (50 ng/ml) (eBioscience) for 18 hrs. Cell supernatants were harvested by centrifugation at 1,500 rpm for 5 minutes and then used for the measurement of IL-22.
In vitro lethal toxin assay with MNK-3 cells:
MNK-3 cells (an ILC3 cell line) were cultured in MNK-3 media (DMEM (High glucose), 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM Glutagro, 1 mM sodium pyruvate, 10 mM HEPES, 55 M -mercaptoethanol, 5 g/ml gentamicin, 10 ng/ml IL-7 and 10 ng/ml IL-15). For lethal toxin experiments MNK-3 cells were cultured in media containing 10 ng/ml IL-7. Cells were treated with lethal toxin for 2 hrs followed by IL-23 stimulation for 6 hrs. Cell lysates were analyzed for RNA and supernatants were analyzed for secreted IL-22 by ELISA.
Lethal Toxin (LT) = PA + LF
• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:
• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence