Shiga Toxin 2 from Escherichia coli

Snippet: ... 37°C with gentle shaking (1000 rpm) for 24 hours before analysis. To determine whether the observed effects were specifically mediated by EhxA rather than Stx, RBCs were treated with purified Stx2 (List Biologicals, 162L) (10). A FACS Calibur system (Becton Dickinson) equipped with a 488-nm argon-ion laser was used for all meas ...

• Product #162L – Shiga Toxin 2 from E. coli

Snippet: Materials and MethodsToxinsStx2a purified from Escherichia coli was purchased from List Biological Laboratories, Inc. (USA) ...

• Product #161 – Shiga Toxin 1 from E. coli
• Product #162L – Shiga Toxin 2 from E. coli

Snippet: motic milieu of the kidney significantly shapes the outcome of toxin-induced injury in HUS. Materials and MethodsToxins and Sodium Chloride (NaCl) Purified Stx2 from Escherichia coli was purchased at List Labs (List Biological Laboratories, INC., USA). 5M Sodium chloride solution was prepared from Biosolution Co. (Biosolution, Republic of Korea) and filtered with a 0.45 micron filter. NaCl was dil

• Product #162L – Shiga Toxin 2 from E. coli

Snippet:

• Product #162L - Shiga Toxin 2 from Escherichia coli

Shiga toxin work

Shiga toxin 2 (Stx2) was obtained commercially from the USA through List Biological Laboratories Inc. (product #162 lot 1621A1). Stx2 was chosen as it is known to be more cytotoxic in mice than Stx1; and because epidemiological data from human studies have demonstrated that Stx2 producing STEC have a stronger association with more severe disease.22 All work involving the toxin was performed in a biological cabinet under category 3 facilities. Prior to any toxin work commencing, a ‘control of substances hazardous to health regulations' (COSHH) risk assessment was completed which was submitted and reviewed by the biological and genetic safety committee.

Upon receipt of Shiga toxin, the lyophilized powder was reconstituted with 100μL distilled water and gently mixed by inversion in-keeping with the manufacturer's instructions. This resulted in a stock concentration of 10μg/100μls which was aliquoted into 20μL stock eppendorfs and stored at −20°C until use. The toxin was assessed for purity on 12% SDS-PAGE and was found to have two major bands at molecular weights of 33 kDa and 8 kDa corresponding to A and B toxin subunits, respectively. Purity was >98% and the endotoxin content determined on kinetic chromogenic LAL assay at List Labs was <90EU/mg. A dose of 10 ng/g of Shiga toxin diluted in sterile normal saline 0.9% (Baxter) was given to each mouse via intraperitoneal injection. All in vitro Shiga toxin cell treatments were performed on differentiated cells at a dose of 0.1 ng/ml.

Snippet:

2.1. Bacterial Strains, Reagents, Growth Conditions, and Antibiotic Susceptibility

... The presence of Stx2 (List Labs, Campbell, CA, USA) was determined by Western slot-blot analysis of filtered culture supernates with an anti-Stx2 monoclonal antibody [57] that was obtained from Dr. Alison D. O'Brian (Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA). The results of these studies and other characteristics [58,59,60] are presented in Table 1.

• Product #162L - Shiga Toxin 2 from Escherichia coli

Snippet:

4.5. Treatment of Stx2 on the Chip

Stx2 (List labs; Campbell, CA, USA) was diluted in the DMEM/F12 as supplemented with 5% (v/v) FBS, 100 unit/mL penicillin, and 100 μg/mL streptomycin. One hundred microliters of Stx2 was added only in the gut module, i.e., after assembly of the gut and kidney modules.

• Product #162L - Shiga Toxin 2 from Escherichia coli