Bacterial Toxin Research Citations

2.2.2. Cell Culture

HEK293 human embryonic kidney cells (gift of Frank Graham) were maintained
in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum
(FBS). This cell line has been transformed with the E1 region of type 5
adenovirus (Graham et al., 1977). NIH3T3 murine embryonic fibroblasts (ATCC
#CRL-1658) were maintained in high glucose Dulbecco’s Modified Eagle Medium
(DMEM) supplemented with 10% FBS. hTERT-HME1 human mammary
epithelial cells (ATCC #CRL-4010) were maintained in DMEM/F12 supplemented
with 10 mM HEPES, 10 µg/mL insulin, 5 mg/mL BSA, 10 ng/mL Cholera toxin, 10
ng/mL epidermal growth factor (EGF), and 0.5 µg/mL hydrocortisone. MCF-10A
human mammary epithelial cells (ATCC #CRL10317) were maintained in
modified DMEM/F12 with 5% FBS, 10 µg/mL insulin, 10 ng/mL Cholera toxin, 20
ng/mL EGF, and 0.5 µg/mL hydrocortisone. MTHJ murine mammary
adenocarcinoma cells were established as described (Hummel, Safroneeva, and
Mossman, 2005) and maintained in high glucose Dulbecco’s Modified Eagle
Medium (DMEM) supplemented with 10% FBS. ZR-75-1 human breast cancer
cells (ATCC #CRL-1500) were cultured in RPMI 1640 supplemented with 10%
FBS. All cell lines were supplemented with 2 mM L-glutamine, 0.1 mg/mL
penicillin, and 100 U/mL streptomycin, and grown at 37ºC in a humidified
atmosphere at 5% CO2. All media were purchased from Sigma Chemicals and
all supplements were purchased from Gibco except for cholera toxin (List
Biologicals).

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

Mice and EAE Induction:

… Lgals1^/ and WT mice were immunized subcutaneously in two sites (left and right flanks) with 150µg of MOG_35-55 peptide that was emulsified in complete Freunds adjuvant (CFA; Sigma-Aldrich, St. Louis) containing 200µg Mycobacterium tuberculosis (Difco, Detroit). Mice received 200 ng pertussis toxin (PT; List Biological, Campbell, CA, USA) in 0.2 ml PBS by intraperitoneal injections at the time of immunization and 48 hr later. …

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)

Toxins:

PA+FP59 was the toxin for cytotoxicity assays. … PA was also purchased from List Biological Laboratories (Campbell, CA).

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

SNAPtide assay of BoNT/A LC with screened compounds:

SNAPtide (o-Abz-Dnp) was purchased from List Biological Laboratories and was used
for analyzing the kinetics of protease inhibition.  The BoNT/A LC is included in a mixture
(denoted Big Mix) to minimize discrepancies in concentration of enzyme and to have a constant
concentration of BoNT/A LC throughout the whole experiment.  Big Mix includes 1xPBS,
20M Zn2+, and 0.2M LC/A-SUMO.   Compounds B, D, and E were dissolved in DMSO and
the LC Buffer (1xPBS, 20M Zn2+) and DMSO was tested as a negative control to determine any
possible interference in fluorescence.  The catalytic activity of BoNT/A LC is largely insensitive
to the presence of DMSO, making this an ideal co-solvent (Boldt et al, 2006).   The SNAPtide
assay was initiated by the addition of 2M SNAPtide and the reaction was allowed to proceed to
5% completion and the initial rate was determined.  The protease activity was monitored by the
increase of donor fluorescence intensity.  The compound 7-(phenyl(8-quinolinylamino)-8
quinolinol, better known as QAQ (84096), has previously shown in an HPLC-based enzymatic
assay to inhibit BoNT/A LC by 74% at 20M enzyme concentration (Lai et al, 2009). …

• Product #520 – SNAPtide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin

EAE Mice Model and Treatment Protocols:

C57BL/6 mice were obtained from the Animal Center of Third Military Medical University. All experiments were performed in accordance with Health Guide for the Care and Use of Laboratory Animals, with the approval of Third Military Medical University Committee on Animal Care (permission NO: SCXK-JUN-2007-015). Female C57BL/6 mice (N=43, 8 weeks old) were immunized subcutaneously with 200 µg of MOG₃₅₅₅ peptide (Invitrogen, Carlsbad, CA) emulsified in complete Freund adjuvant (CFA, Sigma Aldrich, Saint Louris, MO) on Day 0 and Day 7, and received 300 ng pertussis toxin (PT, List Biological Laboratories, Campbell, CA) in 0.1 ml PBS intraperitoneal at the time of immunization and 48 hours later. …

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)

Immunization with Purified Antigens:

As previously described (38), mice were immunized intranasally with 4 µg of recombinant protein and 1 µg cholera toxin as adjuvant (List Biological Laboratories) per 20 µL dose. Control mice received adjuvant alone. Three immunizations were given at weekly intervals, followed by intranasal S. aureus challenge at week 5, as described above.

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

Adjuvants:

B. pertussis toxin and DNA containing unmethylated CpG-dinucleotide were purchased from List Biological and Operon, respectively. NE was made at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA. This adjuvant is composed of a water phase (PBS, Tween 80) and oil phase (Squalene with or without -tocopherol) at a 4:1 ratio. The nanoemulsion was prepared by passing the mixture through M-110 PMicrofluidizer (Microfluidics) under a pressure of 18,000 psi. …

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)

Animal models and ethics statement:

… All immunizations were performed by instilling 20 µl of a mixture of PBS, 1 µg of cholera toxin (CT; List Biologicals, Campbell, CA) and either 4 µg of the indicated purified pneumococcal protein or 100 µg of WCC as immunogens onto the nares of gently restrained unanesthetized mice. Control animals were immunized with CT mixed in PBS alone to control for any nonspecific effect of the adjuvant alone. Animals were immunized intranasally twice at one-week intervals. …

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

… After this, cells were preincubated for 30 min with 50 ng/mL of IL-10, 5PEGIL-10 or 20PEGIL-10 in 0.1 mL FBS-free medium containing 0.5% normal mouse serum. At t = 0, 25 ng/mL of LPS (E. coli, List Biological Laboratories, INC., USA) was added to the wells. …

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/

Materials:

The following TLR agonists were purchased: smooth LPS from E. coli O55:B5 (List Biologicals) …

Macrophage culture and cytokine assays:

BMDMs were washed three times in serum-free medium, followed by incubation overnight with HMGB1, with or without hemoglobin, or with different TLR agonists as desired at indicated concentrations.NOTE:  At the time this paper was written, Product #203 (5mg – LPS from Escherichia coli O55:B5) was utilized.

NOTE:  At the time this paper was written, Product #203 (5mg – LPS from Escherichia coli O55:B5) was utilized.

Product #203 (5 mg – LPS from Escherichia coli O55:B5) is no longer sold.

Product #203A (2.5 mg – LPS from Escherichia coli O55:B5) is available for purchase.

• Product #203A – LPS from Escherichia coli O55:B5