Bacterial Toxin Research Citations

… Diphtheria Toxin (DT) List Biological Laboratories Inc Cat# 150 …

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

… DT, unnicked (List Biological Laboratories), was injected intraperitoneally at a dose of 20 ng per g of body weight in 1 × PBS for initial depletion and phenotyping. Where applicable, DT at a dose of 4 ng per g of body weight in 1 × PBS was then injected intraperitoneally every 3 d for continuous depletion. We used 1 × PBS alone as a vehicle control. …

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

Microglia depletion

For microglia depletion, 80 ng of diphtheria toxin (DT) (0.5 ng/μL, 150, List Labs, USA) was injected intraperitoneally on day -2 and 0 before SAH induction.

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

Diphtheria toxin administration:

For depletion of CD206-expressing macrophages, 500ng (20ng/g body weight, assuming an average 25g weight for each mouse) diphtheria toxin (DTx;List Biological Laboratories) in 100µL 1X PBS was injected intraperitoneally into each mouse – for both Csf1rCre; Mrc1(CD206) LSL-Venus-DTR (DTR) and Mrc1(CD206) LSL-Venus-DTR (WT) groups – at every time point. For the early depletion regime, injections were started 2 days after adoptive transfer of T cells and continued every 2-3 days till endpoint, while for the late depletion regime, injections began at d10 after T cell injection and continued till endpoint. For testing the effects of DTx in tumor-free tissue, similar dosing of DTx as the early depletion regime was implemented without tumor injection, and the skin (ectopic tumor site) and skin-draining lymph nodes were isolated for analysis. Mice were found to be healthy and without frank health issues with 6 doses of 500ng DTx (early depletion regime), but were monitored nevertheless throughout the experiment, as per IACUC guidelines.

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

Immunization and in vivo treatment

All immunization procedures were conducted in the rear footpad with 20 µL injection volume per
footpad. Mice were immunized as indicated in each Figure with 5 µg low-endotoxin OVA (Worthington
Biochemical Corporation) mixed with either 50 µg papain (P4762, Sigma), 10 µL FCA (F5881, Sigma),
or 10 µL alum (77161, Thermo Scientific) in phosphate-buffered saline (PBS). For DC depletion, mice
were injected i.p. with 500 ng DT in PBS (List Biological Laboratories)
at indicated time-points. …

T cell priming assays

… Mice with DTR expression were treated with DT as indicated in each Figure. …

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

… For cell ablation, 1 mg/ml stock solution of DTX (List Biological Laboratories, 150) was prepared with water, aliquoted and stored at -20°C until use. …

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

Materials and Methods

Animals.
Mice of both sexes were used between 4-17 weeks of age and maintained on a C57BL/6
background under specific pathogen-free (SPF) conditions in Allentown caging with aspen shavings and
LabDiet5010 chow (Purina, St. Louis, MO) and RO water. TNFDARE/+ mice8 were rederived by the Genome
Editing Shared Resource, Rutgers Cancer Institute of New Jersey and subsequently crossed to TcrdGDL
mice67. TNF+/+ littermates were used as controls. For suppression assays, wildtype C57BL/6 mice or
TcrdEGFP68 mice were used between 8-14 weeks of age. To assess IEL proliferation, 200 µg of
5-ethynyl2′-deoxyuridine (EdU)(Sigma-Aldrich) was administered i.p. daily for 4 days. To induce gd T cell depletion
in 4-week-old TcrdGDL; TNFDARE/+ mice, 15 ng of diphtheria toxin (List Biological, Campbell, CA) or
endotoxin-free PBS per gram body weight was administered i.p. 24 and 48 h prior to the experiment. …

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

In vivo cell depletions

Animals were depleted of CD4+ or CD8+ T cells as previously described [19,85]. Briefly, animals were treated with 0.5 mg of rat anti-CD4 mAb GK1.5 (Leinco) or 0.2 mg rat anti-CD8 mAb 2.43 (Leinco) in 200 μl of DPBS i.p. one day prior to challenge. Treatment was repeated once weekly for the entirety of each study. For depletion of B cells, animals were treated with 250 μg of rat anti-CD20 mAB MB20-11 (BioXcell) in 200μl of DPBS i.p. one week prior to challenge [86]. Anti-CD20 treatment was repeated on day 14 post infection. Control animals for both T and B cell depletions received equivalent dosages of rat or mouse IgG (Leinco or Southern Biotech) respectively. Upon euthanasia, depletion of splenic CD4+ T, CD8+ T, or B cell populations were confirmed to be ≥ 90% effective via flow cytometry. TReg were depleted in DEREG animals by administering 1 μg DTX (List Biological Laboratories) resuspended in 100 μL DPBS i.p. to each animal on day 14 and 15 post challenge [59]. WT animals were administered an identical dose of DTX as a control. Blood was collected from animals to confirm systemic depletion of FoxP3+CD4+ T cells seven days post treatment (S3A Fig).

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

… Diphtheria toxin (List Biological, catalog number: 150) …

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

… Cat# 555029 Diphtherin List Labs Cat# 150 …

Adoptive transfer of immune cells

For the adoptive transfer of BMDCs, Itgax(CD11c)-DTR/EGFP-recipient mice (age 6–7 weeks, male) were intraperitoneally injected with 100ng diphtherin (DT) and administered with WT BMDC or Slc38a5−/− BMDC cells (1 × 106 cells suspended in 100 μL sterile PBS) via the ophthalmic vein. DT injection and cell transfer were performed every 3 days starting from one day prior to IMQ treatment, and 3 times of DT injection plus cell transfer were performed (Figure S4B). The mice in the control groups received injection of sterile PBS (100 μL). …

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