Diphtheria toxin dtx

All animal experiments were performed according to approved protocols (MO19M266, MO19M366, and MO20M142) of the Animal Care and Use Committee (ACUC) at Johns Hopkins University (JHU). The PDGFRα-CreER^TM animals were a kind gift from the Dwight Bergles laboratory^{56 (link)} and are commercially available (The Jackson Laboratory, Stock No. 018280, Bar Harbor, ME). Pdgfrα^mT/mG mice were obtained by crossing Pdgfrα-CreER^TM with mT/mG mice (JAX Stock No. 007576). Pdgfrα^iDTR;mT/mG mice were obtained by crossing Pdgfrα^mT/mG mice with iDTR mice (JAX Stock No. 007900). Unless otherwise specified, male mice were used in all the experiments. NOD SCID mice were purchased from the Jackson Laboratory (JAX Stock No. 001303). Tamoxifen (TM; Sigma–Aldrich, St. Louis, MO) and diphtheria toxin (DTX; Sigma–Aldrich) were injected intraperitoneally according to previously validated protocols (TM: 150 mg·kg^-1 per day for 5 d; DTX: 45.7 μg·kg^-1 per day for 3 d).^{14 (link),57 (link)} TM was dissolved in sunflower seed oil (Sigma–Aldrich). Post-TM chase periods ranging from 7 d to 9 mo were assessed. When feasible, a littermate analysis was performed by investigators blinded to the mouse genotype.

For depletion of dendritic cells, CD11c-DTR mice were treated with 100ng Diphtheria toxin (Dtx) (Sigma) via an i.p injection, one day prior to experimental immunizations. Upon initial reconstitution of DTx, the DC-depleting efficiency was confirmed by flow cytometry of splenocytes from CD11c-DTR mice which had received DTx i.p one day prior, and the absence of CD11c+ DCs compared to a C57BL/6 mouse which had received Dtx. Macrophages were depleted following i.v injection of clodronate liposomes (Liposoma BV) 8 days prior to immunization as described previously (42 (link)). Depletion of macrophages was confirmed by flow cytometry as shown previously (43 (link)).

Femurs and tibias of donor mice were removed and BM was flushed with cold PBS. BM was washed with cold PBS twice and filtered by 100 μm filter. Cells were suspended with PBS and loaded on equal amount of Ficoll (GE healthcare). Tubes were centrifuged 920 g in room temperature for 20 min without breaks and Buffy coats were collected and washed with cold PBS. CD11c-DTR > wt]. Cells were stained and sorted according to the following markers: CD117^- CD11b⁺ CD115⁺ Ly6C⁺ GFP^int. BM chimeras were treated with 18 ng / gram bodyweight Diphtheria toxin (DTx) (Sigma-Aldrich, Cat # D0564) for two consecutive days before transfer. At the day of transfer mice were injected with 10⁶ BM monocytes IV. At days 1, 3, 5, 7 and 9 after transfer mice were injected with 9 ng / gr bodyweight DTx.

E13.5 WT or E16.5 BDA1; Lgr5^DTR/+ embryonic paws with skin on the ventral side removed were cultured ventral side down on culture inserts (Millicell PICM0RG50, 0.4 μM hydrophilic PTFE membrane) on a 6-well plate. Each insert contains 4-5 paws and wells were filled with a minimal volume of Gibco^TM BGJb medium (Thermo 12591038) just sufficient to moisten the insert membrane. Caspase inhibitor zVAD-FMK (R&D FMK001) and Diphtheria toxin (DTx) (Sigma D0564) were diluted to 20 mM and 2 mg/ml, respectively, according to the manufacturer’s protocol. Culture media was changed daily.