Bacterial Toxin Research Citations

Experimental autoimmune encephalomyelitis (EAE):

Induction of EAE was performed in accordance to (Miller et al., 2007)). In brief, a 1 : 1 emulsion mixture of myelin oligodendrocyte glycoprotein (MOG35-55) (Genemed Synthesis Inc., San Antonio, TX, USA) and complete Freunds Adjuvant (CFA) (Sigma-Aldrich, St-Louis, MO, USA) supplemented with 10 mg/ml of heat-killed Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI, USA) was prepared. Female mice (8-10 weeks old) were injected subcutaneously at two sites (100 l per site) adjacent to the tail with the emulsion. Pertussis toxin (200 ng) (List Biological Laboratories Inc., Campbell, CA, USA) was administered intraperitoneally on the day of immunization. …

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)

… Full-length CtxA1, full-length PtxS1 (List Biologicals, Campbell, CA), or peptide (Peptide 2.0, Chantilly, VA) was amide-coupled well plates were incubated with 1 µg of Ptx (List Biologicals) in serum-free Ham’s F-12 …

Full-length CtxA1 and full-length PtxS1 have been discontinued.

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)

… The emulsion was injected subcutaneously in both hind flanks of each animal. The mice were intraperitoneally -treated with 300 ng of pertussis toxin from bordetella pertussis (dissolved in 100 µl PBS; List Biological Lab, USA) at the time of immunization and again 48 …

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:

On days 0 and 2, 150 ng/mouse pertussis toxin (List Biological Laboratories) was given by intraperitoneal injection. Mice were monitored and weighted daily until day 28. …

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)

… In vivo depletion of Foxp3 + Treg Foxp3 + Treg were depleted from DEREG mice[10] by injection of 500ng Diphtheria toxin (DT) (List Biological Laboratories, Inc., Campbell, CA) and two days later mice were grafted as described below …

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

Toxins:

…  Recombinant EF and PA from Bacillus anthracis were purchased from List Biological Laboratories, Inc., Campbell, CA. …

Signal Transduction Experiments:

Zymosan particles were opsonized by incubating the particles with 50% human serum for 30 min at 37 C. The particles were then washed and dissolved in DMEM with FCS before use for treatment of cells (100 g of zymosan particles per 10⁶ cells). To investigate the effects of toxin-provoked cAMP intoxication on opsonophagocytic signaling, 3 10⁶ THP-1 cells were preincubated with CyaA (150 ng/ml; 0.85 nM) for 30 min or ET (625 ng/ml; 7.04 nM EF + 2500 ng/ml; 30.24 nM PA) for 6 h, respectively (based on cAMP ELISA experiments to have similar cAMP intoxication levels, cf. Figure 1A,B). The cells were then treated with 300 g of serum opsonized or unopsonized zymosan as positive and negative controls for 30 min, respectively.

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #178A – Anthrax Edema Factor (EF), Recombinant from B. anthracis

EAE:

On day 0, 8-week-old mice were immunized subcutaneously with 100 µg MOG35-55 peptide emulsified in complete Freunds adjuvant (Sigma-Aldrich) containing 4 mg/ml Mycobacterium tuberculosis extract (H37Ra; Difco), distributed between the 2 hind flanks. On days 0 and 2, 100 ng/mouse pertussis toxin (List Biological Laboratories) was given by intraperitoneal injection. …

Adaptive transfer EAE using PKM2 inhibitor:

Naive CD4⁺ T cells from 2D2 mice were cultured with 20 µg/ml MOG35-55, 1 10⁶ mitomycin-treated splenocytes, and 0.5 ng/ml rIL-12 for 48 hours as previously described (38, 39) and 0.1 M shikonin or the same concentration of DMSO was added on day 0. Cultured cells were harvested on day 2 of culture. Five million cells were suspended in 150 µl of PBS (pH 7.4) and injected intravenously into each Rag1-deficient mouse. Pertussis toxin (300 ng per mouse; List Biological Laboratories) was intraperitoneally injected later on the day of transfer and 2 days later. Mice were monitored and weighed as previously described (11).

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)

Materials:

…  DT and CT were purchased from List Biological Laboratories (Campbell, CA). …

Intoxication assays:

HeLa or CHO cells grown to ~80% confluency in 24-well plates were challenged with varying concentrations of DT or CT in serum-free medium. Where indicated, cells were also exposed to 20 M PES for 1-2 h or 10 M CysA for 30 min before toxin addition. …

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

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. 

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

Experimental autoimmune encephalomyelitis (EAE) in mice:

For induction of EAE, 810-week-old female mice were immunized with the MOG₃₅₅₅ peptide (100 g/mouse, AnaSpec) in complete Freunds adjuvant (Difco Laboratories). On day 0 and day 2, pertussis toxin (100ng/mouse, List Biological Labs) was injected intraperitoneally. EAE was also induced with adoptive transfer of MOG-stimulated T cells. For this, C57BL/6 mice were immunized with MOG₃₅₅₅ 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)

Cell isolation and culture:

Nave CD4⁺ T cells were isolated from splenocytes and lymph node cells with the naive CD4⁺ T cell isolation kit (Miltenyi Biotec). To obtain CNS APCs, brains from newborn mice were collected, dissociated with scissors and digested with collagenase type 4 (2mg/ml, Worthington Biochemical) for 1hour at 37C. Mononuclear cells in the interphase between 30% and 70% of Percoll (GE healthcare) were isolated by density-cut centrifugation as described³⁸. These cells were cultured for 4 days and then activated by TNF- (10ng/ml, BioLegend) and IL-17 (25ng/ml, BioLegend) in the presence and absence of SCFAs (C2 at 10mM, C3 at 1mM, C4 at 0.5mM) for additional 3 days in DMEM medium (10% FBS). Adherent glial cells, largely composed of microglial cells, were co-cultured as APCs with nave CD4⁺ T cells at 1:10 ratio for 56 days in the presence of Staphylococcal enterotoxin B (SEB, 5 g/ml, List Labs) to stimulate nave CD4⁺ T cells isolated from the spleen and lymph nodes of unimmunized C57BL/6 mice.

… The column was washed with T205N100P0.1 followed by T205N100P0.1 plus 20 mM imidazole and eluted with T205N100P0.1 with 150 mM imidazole. Peak fractions were pooled and diluted tenfold with T20P0.1, loaded onto a Source Q anion exchange column, and eluted by a 0-1M NaCl gradient. Purified EF was concentrated to approximately 20 mg/mL and frozen at -80C. Protein quantitation was performed using extinction coefficients calculated from the known primary sequence of each protein on the Expasy ProtParam webserver. PA was purchased from List Labs. …

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis