CNV was induced as previously described (51 (link)). Briefly, mice were anesthetized and eyes were dilated and lasered with a slit lamp (Zeiss) mounted ophthalmic laser (IRIDEX). Each eye was treated with either 4 (CNV area quantification) or 8 (scRNA-Seq and flow cytometry analysis) focal laser burns (75 μm, 110 mW, 100 msec). Eyes with major bleeding at the time of laser were excluded. In Mac-depleting CD11c+MaciDTR mice, we i.p. injected 4 ng DT/g (MilliporeSigma, D0564, dissolved in PBS) at the time of laser injury and for every 2 days thereafter.
D0564
Manufactured by Merck Group
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D0564 is a laboratory equipment designed for general use in various research and analytical applications. It serves as a multipurpose device for tasks such as stirring, mixing, and agitating samples. The product specifications and technical details are available upon request.
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Lab products found in correlation
Bp0075 1, by BioXCell (2 mentions)
Bp0089, by BioXCell (2 mentions)
P 1000 micropipette puller, by Sutter Instruments (1 mentions)
T5648, by Merck Group (1 mentions)
Jes6 1a12, by BioLegend (1 mentions)
Recombinant mouse il 33, by BioLegend (1 mentions)
Recombinant mouse il 2, by Thermo Fisher Scientific (1 mentions)
L2630, by Merck Group (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
F70101c n, by Formumax Scientific (1 mentions)
Rs 2000 pro, by Rad Source (1 mentions)
Nanoject 3 injector, by Drummond (1 mentions)
Ms peg 4 methyl peg nhs ester, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 2000 uv visible spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Pd miditrap g 25 column, by GE Healthcare (1 mentions)
C57bl 6, by RIKEN BioResource Center (1 mentions)
Ap20187, by Takara Bio (1 mentions)
27 protocols using d0564
1
Laser-induced Choroidal Neovascularization
2
Diphtheria Toxin-Induced Retinal Injury
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Procedures regarding the diphtheria toxin injection timing and concentration followed the method previously described (Hillier et al., 2017 (link); Kim et al., 2020 (link)). Mice were anesthetized for intraocular injection using a mixture of ketamine (80 μg/kg) and xylazine (5 μg/kg). Glass micropipettes for injection were prepared using a P1000 micropipette puller (Sutter Instruments). Injection was performed using a Nanoject III precision pipette (Drummond Scientific). DT (D0564, Millipore Sigma) was diluted to a working concentration of 0.8 ng/μl, and 2 μl was injected into the intravitreal space of each eye twice, 48 h apart. For some mice, 2 μl DT solution at a concentration of 5 ng/μl was injected to the intravitreal space only once. Subsequent behavioral and cellular experiments were conducted 6–10 d after the initial DT injection. Control mice were injected with 0.9% normal saline in both eyes on the same schedule as toxin-injected mice.
3
Neutrophil Function in Liver Transplantation
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We used a mouse model of ex vivo hepatic cold storage followed by liver transplantation (4 (link)). To focus on recipient-derived CC1 neutrophil function while avoiding host alloimmune responses, donor livers (WT) stored in UW solution (4°C/18 hours) were transplanted to syngeneic mouse recipients. Liver graft and serum samples were collected at 6 hours after reperfusion, the peak of hepatocellular damage in this model. The sham-treated group underwent the same procedures except for OLT.
We used a DT-inducible neutrophil depletion (PMNDTR) transgenic mouse model (20 (link)). PMNDTR mice were pretreated with DT (500 ng/mouse, i.p., D0564, MilliporeSigma), and 24 hours after native PMN depletion (assessed by FACS), neutrophils isolated from WT or CC1-KO donor mice were adoptively transferred into PMNDTR mice (3 × 107 cells/mouse, i.v.), followed by warm hepatic ischemia insult (90 minutes) as described (69 (link)). Some PMNDTR mice were pretreated with CI-amidine (50 mg/kg, s.c.; 10599, Cayman Chemical) at 1 hour prior to the ischemia insult. Blood and liver tissue samples were collected at 6 hours after reperfusion.
We used a DT-inducible neutrophil depletion (PMNDTR) transgenic mouse model (20 (link)). PMNDTR mice were pretreated with DT (500 ng/mouse, i.p., D0564, MilliporeSigma), and 24 hours after native PMN depletion (assessed by FACS), neutrophils isolated from WT or CC1-KO donor mice were adoptively transferred into PMNDTR mice (3 × 107 cells/mouse, i.v.), followed by warm hepatic ischemia insult (90 minutes) as described (69 (link)). Some PMNDTR mice were pretreated with CI-amidine (50 mg/kg, s.c.; 10599, Cayman Chemical) at 1 hour prior to the ischemia insult. Blood and liver tissue samples were collected at 6 hours after reperfusion.
4
Granulocyte Depletion Strategies in Mice
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For diphtheria toxin-mediated granulocyte ablation, we treated mice with daily intraperitoneal injections of diphtheria toxin (D0564, Sigma) (0.25µg/mouse/day) for one week starting one week after transplantation. For αLy6G-mediated depletion experiments, we treated mice with 100µg (intraperitoneal) of αLy6G (clone 1A8, BP0075-1, Bioxcell) or isotype control (clone 2A3, BP0089, Bioxcell) at days 1, 3 and 5 after transplantation. Binding of αLy6G prevents staining with αGr1. This prevented the use of Gr1 for detection of granulocytes in αLy6G-injected mice. Thus, in these experiments, Ly6G+ granulocytes were detected by staining BM cells with isotype or αLy6G antibodies: 1µg/ml of αLy6G or isotype antibody was used , followed by staining with a secondary antibody (405418, Biolegend)
5
Tracing Intestinal Stem Cell Lineages
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Lgr5Dtr-GFP (MGI:5294798) (Tian et al. 2011 (link)), R26RTom (MGI:3813512) (Madisen et al. 2010 (link)), Ngn3Cre/ERT2 (MGI:5449724) (Li et al. 2012 (link); Li et al. 2019 (link)), and Lgr5-EGFP-IRES-creERT2 (MGI:J127123) (Barker et al. 2007 (link)) mice were maintained on a predominantly C57BL/6 background. Animals were housed under specific pathogen-free conditions in 12-h light/dark cycles at 23°C ± 1°C and 55% ± 15% humidity. Food and water were provided ad libitum. Animals were weaned 21–28 d after birth and handled and euthanized according to procedures approved by the Animal Care and Use Committee of the Dana-Farber Cancer Institute. Mice were at least 8 wk old at the time of experiments and cell isolations. Mice of both sexes were used in the experiments, with littermate controls. Ngn3-Cre;Lgr5Dtr;R26Tom mice were injected with two daily doses of intraperitoneal tamoxifen (TAM; 50 mg/kg; Sigma T5648), and whole SI was harvested 12, 24, 48, and 72 h after the second dose (Supplemental Fig. S1A ). To ablate ISCs, mice received one dose of intraperitoneal diphtheria toxin (DT; 50 µg/kg; Sigma D0564) at the same time as the second TAM injection.
6
Regulatory T cell regulation in autoimmunity
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DT (D0564; Sigma-Aldrich) was injected i.p. into Foxp3DTR+ and Foxp3DTR− littermates at 30 ng/g of body weight. The regimens for DT administration are depicted in the corresponding figure. For the adoptive transfer of T reg cells into Foxp3DTR+ perinates, 0.4 × 106 TCRβ+CD4+Foxp3+ cells from lymph nodes of 20- to 25-d-old Aire+/+ or Aire−/− Foxp3-IRES-GFP mice were transferred into 8-d-old recipients. For the treatment of Aire+/+ mice with IL-2–αIL-2 mAb complexes, recombinant mouse IL-2 (PeproTech) was incubated with an αIL-2 mAb (JES6-1A12; BioLegend) at room temperature for 10 min before the injection. Mice were injected with 0.02 ml cytokine–antibody complexes containing 2.5 µg/ml IL-2 and 82.5 µg/ml αIL-2 mAb. Recombinant mouse IL-33 (BioLegend) was injected at 12.5 µg/kg; control mice were injected with an equal volume of PBS. αPD-1 mAb (29F.1A12) was injected at 3 mg/kg; control mice were injected with an equal amount of an isotype-matched control mAb (2A3; both from BioXCell). Measurement of blood glucose levels in mice treated with αPD-1 or IL-33 was performed on blood from the lateral tail vein.
7
Inducible Depletion of NG2 Glia and LPS Response
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Cell-type-specific Cre mice were crossed to generate with inducible diphtheria toxin receptor (iDTR) mice to allow expression of DTR in the targeted cell populations. The DTR is expressed after Cre recombinase removes the STOP cassette, increasing susceptibility of specific cell type to diphtheria toxin (DT; Sigma-Aldrich, D0564). Their littermates bearing Cre recombinase were also administrated with DT and used as control. No specific adverse side effects of DT were observed when administered to the control and iDTR mice.
For NG2 glia ablation paradigm, DT administration was performed as described previously with some modifications [30 (link)]. In brief, adult mice (2~6 months old) were administrated with seven intraperitoneal injections of DT (150 ng/mouse) at 12-h intervals (i.e., for 3.5 consecutive days in total). This administration paradigm ensures efficient and specific ablation of NG2 glial cells, given that depletion of NG2 glia is usually followed by a fast repopulation of the cells [20 (link), 31 (link)]. Twelve hours after the final DT injection, mice received single intraperitoneal injection of LPS (2.5 mg kg−1, Sigma-Aldrich, L2630). Animals were sacrificed 4 h later.
For NG2 glia ablation paradigm, DT administration was performed as described previously with some modifications [30 (link)]. In brief, adult mice (2~6 months old) were administrated with seven intraperitoneal injections of DT (150 ng/mouse) at 12-h intervals (i.e., for 3.5 consecutive days in total). This administration paradigm ensures efficient and specific ablation of NG2 glial cells, given that depletion of NG2 glia is usually followed by a fast repopulation of the cells [20 (link), 31 (link)]. Twelve hours after the final DT injection, mice received single intraperitoneal injection of LPS (2.5 mg kg−1, Sigma-Aldrich, L2630). Animals were sacrificed 4 h later.
8
Eosinophil Depletion in iPHIL Mice
9
Depleting IRF4-expressing T Cells in Tumor Models
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DT (D0564, Sigma-Aldrich) was dissolved in PBS and used to deplete IRF4-expressing T cells in tumor-bearing Irf4GFP-DTR mice. In the TRAMP-C1 implantation model, Irf4GFP-DTR mice were s.c. injected with 2 × 106 TRAMP-C1 prostate cancer cells on day 0 and intraperitoneally injected with 25 μg/kg of body weight DT or 100 μl of PBS vehicle on days 20, 21, 22, 40, 41, and 42. In the B16F10 implantation model, Irf4GFP-DTR mice were injected with 0.1 × 106 B16F10 cells on day 0 and intraperitoneally injected with 25 μg/kg DT or 100 μl of PBS vehicle on days 10, 12, and 14. Tumor growth curve and survival rate were determined. In the B16F10 model, the effects of DT administration on TILs were examined through flow cytometry analysis on day 22 after tumor implantation.
10
Conditional Microglial Depletion in Mice
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We used a CX3CR1CreERT2/+:R26iDTR/+ transgenic mouse line for global microglial depletion. Mice were administered tamoxifen (T5648, Sigma) to initiate DT receptor expression (100 mg/kg, gavage twice, with 2-day intervals) at 4 weeks before depletion onset. Microglia labeled with DT receptor were depleted following i.p. injections of DT (D0564, Sigma) for 3 consecutive days (25 μg/kg).
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