Citations

… Mice were injected via tail vein with 10mg/kg LPS (List Labs) as well as 10mg/kg of the pan-caspase inhibitor ZVAD-FMK (BD Biosciences) in order to inhibit apoptosis and sensitize cells to necroptosis as previously described [16, 3235]. …

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/

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.