Citations

Drug administration:

…In other cases they were treated with both flumazenil (10 M) and/or botulinum toxin B (5 nM) across a time-course (1, 2, 4, 6 or 24 h). In all cases, cells were harvested at the end of the treatment so that all groups would be processed in parallel (Inset, Fig. 2). Other drugs used included …and botulinum toxin B was from List Biological Laboratories, Inc. (Campbell, CA).

Reagents and Materials:

All reagents purchased were of the highest grade possible. … The BoNT-A holotoxin was purchased from List Biological Laboratories Inc., Campbell, CA, USA. …

BoNT-A Sera Spike and Dilution Series:

A BoNT-A holotoxin stock was diluted to 1 g/mL in saline to make a working solution. A pool of sera representing each animal species was spiked initially to 100 ngmL¹ from the BoNT-A working solution and serial dilutions using the corresponding animal sera were used to make the dilutions used in the assay titration.

Mice and immunizations:

Adult mice (aged between 7 and 13 weeks) were immunized…and 10 µg CT (List Biological, Campbell, CA) in 200l phosphate-buffered saline (PBS) using a feeding needle that was placed through the mouth into the stomach.

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

Immunofluorescence labeling:

…We used similar double-immunofluorescence protocols to label CTb with FoxP2 or c-Fos. In a 1:3 series, CTb was labeled using a goat polyclonal antiserum (1:5,000; List Biological Laboratories, no. 703, lot no. 115037) in combination with rabbit anti-c-Fos. …

• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Reagents:

Wild-type LPS from E. coli K12 was obtained from List Biological Laboratories (Campbell, Calif). …

Results:

Wild-type LPS stimulated CD14-independent TNF- and IL-10 secretion

We next set out to investigate whether alteration of the O-Antigen affects interactions between CD14 and the LPS molecule. TNF- and IL-10 secretion was measured in PBMCs pretreated with blocking antibody to CD14 and stimulated with 100 ng/mL LPS, RF5, or DPL. CD14 blockade resulted in significant inhibition of TNF- and IL-10 secretion from cells stimulated with 100 ng/mL wild-type LPS, as has been previously demonstrated ⁽¹⁴⁾, but modest amounts of cytokine secretion did occur. With attenuation or removal of the O-Antigen, secretion of both cytokines in the presence of CD14 antibody decreased to control levels (Fig. 5). DPL, even in 1000 ng/mL concentrations, was unable to stimulate secretion of TNF- in the presence of CD14 blockade (data not shown).

• Product #314 – LPS from Escherichia coli K12 strain LCD25

… After 30 min, monocytes were treated with 0.1 ng/ml LPS (S. minnesota R595, List Biological Laboratories, Campbell, CA, USA)

• Product #304 – LPS from Salmonella minnesota R595 (Re)

… We used smooth-type LPS from E. coli O55: B5 (List Biological Laboratories, CA), Rc-type LPS from E. coli J5 (List Biological Laboratories), …

• Product #203 – LPS from Escherichia coli O55:B5
• Product #301 – LPS from Escherichia coli J5 (Rc)

Materials:

…Lipopolysaccharide (LPS) was obtained from List Biological Laboratories (USA). …

Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (USA) at passage 1 and maintained in EBM2 Bullet kit growth medium at 37C in a humidified atmosphere of 5% CO2. In all experiments, cells were used at passage 4~9. HUVECs were plated at 90~95% confluence treated for 1 h with 1, 10, and 20 M of quercetin prior to LPS (1 g/mL) stimulation for 1 or 8 h. …

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/

… For activation of monocytes, 200-L PBMCs were incubated with 10 g/L ultrapure lipopolysaccharide (LPS) (List Biological Laboratories, Inc, Campbell, CA, USA) in separate 96-well plates. …

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/

IFN- neutralization assay:

The ability of IFN- to upregulate LPS-induced production of TNF- by PBMCs was used to test for the neutralizing activity of sera containing high-titer blocking antibodies to IFN-.

AntiIFN-positive sera, negative control sera, or FBS were preincubated for 30 minutes in a 96-well plate (Corning, Corning, NY) at a dilution of 1:5 in RPMI 1640…or LPS (1 g/mL; LIST Biologicals, Campbell, Calif) alone or both stimuli were added.

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/