Citations

In vivo cellular depletion:

For cDC depletion, 25 ng/g of diphtheria toxin (DT; List Biological Laboratories Inc.) was administered intraperitoneally (IP) into the chimeric CD11c-DTR mice. Mice were sacrificed 12 hours later and bone marrow, blood, liver, spleen and lung were harvested for further analysis. For pDC depletion, mice were injected IP with 500 g 120G8 every other day for 14 days. 120G8 and control antibody were purchased from Imgenex. Mice were sacrificed 24 hours after the last injection and livers were harvested. For Foxp3+CD4+T cell depletion, DT was injected IP at a dose of 50ng/g for 2 consecutive days into Foxp3-DTR mice. Mice were sacrificed 48h after the last dose.

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

DT/Diphtheria Toxin Mutant Administration:

CD11b-DTR mice were treated with DT, 15 ng/g body weight (List Biological Laboratories, Campbell CA), by intraperitoneal injection. Control CD11b-DTR mice received the same amount of mutated DT (DTm) (List Biological Laboratories), which does not bind to the DTR. For the current study, we determined the optimal dose schedule of DT administration that could be safely used to evaluate skeletal muscle regeneration allowing long-term survival of the mice. Mice were divided into groups and received one dose of DT from 15 to 35 ng/g body weight in 5 ng/g steps in dosages between groups. The higher DT dose group (range, 20 to 35 ng/g body weight) resulted in 75% mortality at days 7 to 12 after DT injection. By contrast, mice in the low-dose DT group (15 ng/g body weight) had a mortality rate of 17% and could therefore be used in experiments requiring a 21-day time point. To determine whether multiple doses could be used, a cohort of mice was given two to three doses of DT (10 to 15 ng/g body weight) at least 1 week apart. Most of these mice died at days 7 to 12. Therefore, we chose a single dose of 15 ng/g body weight DT to ablate CD11b⁺ cells. These conditions allowed for the survival of the animals through the course of the experiment (21 days) while temporarily ablating the monocyte/macrophage population within a specific time frame. Previous work and our results in the kinetics of DT ablation of monocyte/macrophage populations in multiple tissues including regenerating skeletal muscle and blood indicate that ablation occurs within 12 hours, lasts 24 hours, with recovery generally occurring 48 hours after DT administration.^{7 ;  15} Single injections of DT were administered at various times (0.5, 0, 1, 2, and 4 days) relative to the injection of CTX to transiently ablate monocyte/macrophages at different time points during skeletal muscle regeneration.

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae
• Product #149 – Diphtheria Toxin CRM₁₉₇, Mutant

Mouse models and treatments:

…DT (6.25 ng/g, IP, List Biological Laboratories) was injected at either P1 or P6 into Pou4f3^DTR/+ mice.

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

… Such micropipettes were filled with a solution of cholera-toxin B subunit (CTB, 2% in distilled water; List Biological Laboratories, Campbell, CA, USA) …

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

Tracer injections:

Animals were injected intraperitoneally (i.p.) with a mixture of 45% ketamine (100 mg/ml), 35% xylazine (20 mg/ml), and 20% saline at a dose of 0.16 ml/100 g body weight. Several minutes later, they were placed into a Kopf stereotaxic instrument and injected in the VTA on one side of the brainstem with the subunit of cholera toxin (Ct; List Biological Laboratories, Campbell, CA). Ct (10%) was dissolved in 0.1 M Sorensons phosphate buffer (SPB; pH 7.4) and injected iontophoretically from a 1.0-mm filament-containing borosilicate glass pipette pulled to a diameter of 20 m. The injections were made over a period of 15 minutes using positive pulses of 3 A (7 seconds on/7 seconds off). …

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

Anatomical studies

Retrograde tracer nanoinjection:

In a separate group of rats (n = 5), IHC was performed to determine if PVN presympathetic neurons express NPY Y1R, thereby providing a potential substrate for their direct inhibition by NPY. These neurons were identified following RVLM nanoinjections of the retrograde tracer cholera toxin subunit b (CTb; List Laboratories, Campbell, CA, USA).

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

Reagents and Treatment:

Ultrapure LPS (Escherichia coli 0111:B4) was purchased from List Biological Laboratories (Vandell Way, CA). …

Results:

LPS Stimulates an Autophagy-Like Process in Liver and Hepatocytes – 

We first wanted to determine whether LPS induced autophagy in hepatocytes via TLR4 activation. To do this we investigated LC3II expression and puncta formation, a standard method of assessing autophagy [27], in liver and hepatocytes at time points after LPS treatment in vitro and in vivo. Wild type C57BL/6 (WT) mice were injected intraperitoneally (IP) with LPS (5mg/kg), and livers were harvested after 6 or 24h. Whole cell liver lysates then underwent immunoblot analysis. We found that LC3II protein expression in liver increased significantly compared with baseline (0h; no LPS) and peaked at 6h (Figure 1(a)) suggesting that autophagy increased in liver after LPS in vivo. Similarly, primary hepatocytes isolated from WT mice and treated with LPS (100ng/mL) in a time-course up to 24h also showed increased LC3II protein expression over time (Figure 1(b)) suggesting LPS-mediated upregulation of autophagy in hepatocytes in vitro. …

• Product #421 – ULTRA PURE LPS from Escherichia coli O111:B4

In vitro interleukin (IL)-1 secretion from BMDMs:

BMDMs (110⁵/well) were seeded into 48-well plates and cultured overnight. Then cells were primed with 10 ng/mL lipopolysaccharide (LPS; List Biological Laboratories, Campbell, CA). Five hours later, cells were stimulated with the indicated dose and size of silica particles or with 1 mM ATP (Wako, Osaka, Japan) for four hours at 37C. …

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/

… Unilateral intraocular of 70 l of an aqueous solution of CTb (List biological Laboratories, Inc., Campbell, CA) to 5%, containing sulfoxide (DMSO) to 10% with… CTb Goat [1:5000] List biological  …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

The Anthrax Lethal Factor was obtained from List Biological Laboratories, INC.

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).