Bacterial Toxin Research Citations

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

Cell culture:

The human monocytic leukemia cell line THP-1 (Cell Lines Service GmbH) was cultured in RPMI supplemented with 10% fetal calf serum (FCS, Life Technologies, Basel, Switzerland) and used below passage 10. THP-1 cells were differentiated into macrophages in the presence of 50 ng/ml phorbol myristate acetate (PMA, Sigma, USA) for 48 hours. THP-1 PMA-derived macrophages were stimulated with 10 ng/ml E. coli LPS (List Biological Laboratories, California) for 2 hours. …

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

Materials and Methods

Anthrax lethal factor (Product #172 or #169), are products of List Biological Laboratories, Inc. The 96-well, black, flat bottom, non binding plates used for the fluorescent plate assay were from Corning (cat # 3991). Bovine plasma (cat # 7310806) was
purchased from Lampire Biological Laboratories.

Sample Preparation: Stock solutions of the fluorogenic substrates, MAPKKide® Plus (± biotin), were made 2.5 mM in
DMSO based on the peptide content determined by elemental analysis. The substrates were diluted in assay buffer: 20 mM
HEPES, pH 8.0 containing 0.1% Tween-20.

LF Activity Assays:
A rapid microplate assay method was evaluated for two ranges of LF: 10 to 1000 pg LF/ml 1:10 diluted bovine plasma and 5 to 250 ng/ml 1:5 diluted bovine plasma. Dilution of the bovine plasma was necessary in order to minimize background.
For the method detecting higher levels of LF (5 to 250 ng/ml 1:5 diluted bovine plasma), 10 μM MAPKKide Plus was added directly to the 1:5 diluted bovine plasma, and the assays were run at 37°C using the kinetic mode of the plate reader with
readings at 1 or 3 minute intervals. The samples were run in triplicate with 9 replicate blanks. The limit of detection was
calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 9).
For the range 10 and 1000 pg/ml 1:10 diluted plasma, 1.25 μM MAPKKide Plus was added directly to the diluted bovine
plasma and the time-dependent increase in fluorescence was monitored at 37°C hourly for 5 hours. For the blank samples
containing no LF there were 3 sets of quadruplicates and the standard deviation was calculated from these three sets. At
each time point, the plate was read 5 times to increase the precision of the fluorescence readings. The standard curve was
analyzed using a linear regression fit forcing the intercept through the mean value of the blanks. The limit of detection was
calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 3 sets of quadruplicates) and calculated as
pg LF/ml plasma using the standard curve. Subsequently, 6 data sets were evaluated, 2 data sets per day for 3 consecutive
days. The data is presented as the average of these 6 data sets, each with 4 replicate samples and 12 replicate blanks. At
each time point, the plate was read 3 times to increase the precision of the fluorescence readings.

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

… A total of 200 ng of Pertussis Toxin (List Biological Laboratories) was given ip on day 0 and 2. …

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)

… Samples were challenged with 10 ng mL-1 of lipopolysaccharide (LPS, List Biological Laboratories inc.) and treated with PBS or increasing concentrations of AZ6983, 0.1, 1, 10 or 100 mol L-1. …

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/

Mouse immunization:

Fresh leaves were crushed while frozen and mixed in distilled water. The quantity of leaf matter administered to each mouse was adjusted to deliver 0.2 µg of AO, and it was mixed with or without CTB (List Biological Laboratories, U.S.A.) (1 µg per mouse). …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt

… and the retrograde tracer cholera toxin subunit b (CTb; low salt, 1% in distilled water, List Biological Laboratories Inc., Campell, CA) were used …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt