Bacterial Toxin Research Citations

LPS:

Ultrapure-grade LPS prepared from Escherichia coli 0111:B4 (List Biologicals, Campbell, CA) was reconstituted in sterile saline at 10g/ml and stored in aliquots at 20C until use in experiments. The specific bioactivity for the individual lot (4216A2) used for this work was not available, but the predicted activity provided by the vendor based on a similar lot was 6.7510⁶ EU/mg (communication from List Biologicals).

In vitro LPS challenge:

Whole blood was collected from animals into sodium heparin tubes (Beckton Dickinson, Caanan, CT) to prevent coagulation. Aliquots (200l) of each blood sample were dispensed into 96-well polypropylene plates and placed at 37C and 5% CO₂ for 30min. LPS diluted in sterile saline or saline only (control) was added to sample wells (10l volume) to achieve final LPS concentrations ranging from 0 to 10g/ml. The plates were incubated for an additional 5h at 37C and then centrifuged at 4C for 10min at 600g to pellet the cells. Plasma collected from each well was stored at 80C until analyzed. The concentration of TNF in each plasma sample was measured by ELISA using the Monkey TNF ELISA kit (U-CyTech, Utrecht, the Netherlands) according to manufacturer instructions. All samples were measured in duplicate.

In vivo LPS challenge and antibody-based modulation:

Animals (n=3/group) were injected IV with 1.0g LPS/kg in a volume of 0.1ml/kg. The LPS was first administered on three different days each separated by a 3-week rest period to establish baseline LPS responses. Prior to the third LPS challenge, blood samples were collected to perform the in vitro LPS challenge (described above) in order to compare in vitro cytokine responses to those induced in vivo. Following another 3-week rest period, animals were injected IV with 0.1, 1, or 10mg/kg of anti-IL-10R1 mAb or with 10mg/kg of the isotype control mAb 2h before a fourth LPS challenge was administered IV. An additional set of control animals was given 10mg/kg anti-IL-10R1 mAb IV and left unchallenged (no LPS). After another 3-week rest period, a fifth and final LPS challenge was performed on all groups.

Blood samples were collected into serum separator tubes (Greiner Bio-One, Monroe, NC) just prior to and 1, 2, 4, 6, and 24h after each LPS challenge to measure concentrations of cytokines (all timepoints) and C-reactive protein (CRP) (pre-challenge and 24h post-challenge only). Blood samples were also collected just prior to and 0.25, 2, 7, 24, 30, 48, 72, 144, 240, 384, and 552h immediately after antibody administration to measure serum anti-IL10R mAb levels. Sera prepared from the blood samples by centrifugation were stored at 80C (for cytokine and CRP assays) or at 20C (for antibody assay) until thawed and analyzed.

• Product #421 – ULTRA PURE LPS from Escherichia coli O111:B4

… LPS was obtained from List Biological Labs (Campbell, USA). …

Author did not specify which List Labs LPS product was utilized in their research.  List Labs provides the following LPS products:  https://listlabs.com/product-information/lipopolysaccharides/

Serological analysis of B. pertussis specific antibodies:

Serological responses specific to B. pertussis antigens were quantified by ELISA. Highbinding microtiter plates were coated with PT (50ng/well)(LIST Biologicals) and FHA (50ng/well)(Enzo Life Sciences), as described in Boehm et al. 31 Serological responses against UT25Sm1 were cultured to an OD600 of 0.24 and microtiter plates coated with 50 µl of bacteria per well. Bound antibodies were detected using goat anti-mouse IgG, IgA, IgG2a, and IgG1 antibody conjugated to alkaline phosphatase (Southern Biotech). Positive antibody titers were determined using a baseline set at two times the average of blanks. 

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

EAE induction and scoring:

NOD and IgH[MOG] mice were immunized subcutaneously with 200µg MOG_[35-55] (Feldan), emulsified in incomplete Freunds adjuvant (BD Difco) that was supplemented with 500µg M. tuberculosis extract (BD Difco). On day 0 and day 2 post-immunization, mice received 200 ng pertussis toxin (List Biological Laboratories) intraperitoneally. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

… Female C57BL/6N mice were immunized for active induction of EAE as our previous described [26]. Briefly, the mice were subcutaneously injected with 200µg MOG3555 in complete Freunds adjuvant (5 mg/ml, Sigma-Aldrich). Pertussis Toxin (200 ng, List Biological Laboratories) was injected intravenously twice on 0 and 2 days postimmunization (dpi.). …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

EAE model:

… On days 0 and 2 of disease course, mice were also injected IP with 375ng of pertussis toxin (List Biological Labs, Campbell, California, USA). Mice were sacrificed at peak disease, PID 16 and late stage, PID 42. For controls the same protocol was used but mice were immunized with CFA without peptide. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

… iDTRwt/ki mice were injected for three consecutive days intravenously with 0.5 g of unnicked Diptheria Toxin from Corynebacterium diphtheria (List Biological Laboratories) diluted in sterile PBS. On day 4 niche cell depletion was analyzed by flow cytometry …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

… pertussis toxin was purchased from List Biological Laboratories (Campbell, CA); …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

… For Foxp3+ cell depletion in DEREG mice, diphtheria toxin (#150; List Biological Laboratories, Campbell, Calif) was administered intraperitoneally 12 hours before each Cl2 exposure at a concentration of 40 ng/g body weight. …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

Experimental DesignTolerance Induction, Sensitization and Challenges:

… Following this oral tolerance induction period, mice were sensitized i.g. once a week for five weeks to the hens egg protein ovalbumin (OVA; 20 mg/0.5 mL PBS; grade V; Sigma-Aldrich, Zwijndrecht, The Netherlands) while using 10 µg cholera toxin (CT; List Biological Laboratories, Campbell, CA, USA) as an adjuvant (days 0, 7, 14, 21, 28; n = 8/group). Sham-sensitized control mice (n = 6) received CT alone (10 µg/0.5 mL PBS). …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae