Zirconium-89 (t1/2 = 78.4 h) was either purchased from 3D Imaging, LLC (Maumelle, AR) or produced at Memorial Sloan Kettering Cancer Center on a TR19/9 cyclotron (Ebco Industries Inc., Richmond, BC, Canada) via the 89Y(p,n)89Zr reaction and purified as previously described.37 (link) 1.0 M sodium carbonate was used to bring the 89Zr-oxalate solution to neutral pH. The neutralized 89Zr solution (100 μCi) was then added to the PNP solutions, brought to 70 °C at 500 rpm on an Eppendorf thermomixer, and incubated for 1 h. To determine the radiochemical yield (% of radioactivity bound to the PNPs, 1 μL samples were taken from samples for instant thin-layer chromatography (ITLC) analysis, using silica-gel-impregnated ITLC paper (Agilent Technologies, Santa Clara, CA). The mobile phase was 50 mM EDTA (pH 5.5) with analysis on a Bioscan AR-2000 radio- TLC plate reader. Unbound 89Zr travels with the mobile front, while bound 89Zr is immobilized at the origin. Radiochemical yield was determined by drawing regions of interest over the origin (bound 89Zr) and comparing this to the total signal. Serum stability experiments were performed at 37 °C in 50% fetal bovine serum (FBS, Gemini Bioproducts), 50% H2O (total volume = 150 μL) or 50% mouse serum (EMD Millipore), 50% H2O (total volume = 150 μL). Samples were analyzed as described for radiochemical yield.
Mouse serum
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
Mouse serum is a blood-derived product obtained from the clotted blood of mice. It contains a complex mixture of proteins, hormones, and other biomolecules that are naturally present in the mouse circulatory system.
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Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Ghost dye red 780, by Cytek Biosciences (2 mentions)
Pe anti human cd279 pd 1 eh12.2h7, by BioLegend (2 mentions)
Rabbit anti sheep biotinylated secondary antibody, by Vector Laboratories (2 mentions)
Ceruloplasmin colorimetric activity kit, by Thermo Fisher Scientific (2 mentions)
Canto 2, by BD (2 mentions)
Anti hla dr, by BioLegend (2 mentions)
Anti cd86, by BD (2 mentions)
Anti cd83, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Goat serum, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Ifn γ, by Abcam (2 mentions)
Duoset elisa kit, by R&D Systems (2 mentions)
Zombie aqua, by BioLegend (2 mentions)
Rat serum, by Merck Group (2 mentions)
Fugene 6 transfection reagent, by Promega (2 mentions)
Protease inhibitor, by Promega (2 mentions)
Formic acid, by Thermo Fisher Scientific (2 mentions)
83 protocols using mouse serum
1
Radiolabeling of PNPs with Zirconium-89
2
Radiochemical Stability Evaluation
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Stability in PBS was determined by incubating 20 µL of the labeled compounds (∼11 MBq) in PBS (80 µL) at 37 °C. Radiochemical purity was determined by iTLC at 30 min, 1 h, 2 h and 4 h. Stability in the serum was carried out by adding 70 µL of the labeled compound (∼35 MBq) to 330 µL of mouse serum (Merck, Haarlerbergweg, The Netherlands). The mixture was incubated at 37 °C. At different time points (30 min, 1 h, 2 h and 4 h), an aliquot of the mixture (50 µL) was added to 50 µL ACN. The vial was vortexed and centrifuged at 10,000 rpm for 20 min and the supernatant was analyzed by iTLC.
3
Affinity Maturation of p19 through Yeast Display
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Affinity maturation of p19 was performed using standard yeast surface display techniques as previously described (18 ) with select modifications. P19 was mutagenized by error-prone PCR and displayed on the surface of yeast fused to human Fc (IgG1). The resulting library was screened by fluorescence activated cell sorting (FACS) following kinetic sorting methods (19 (link)) where clones are selected for a slower dissociation rate. Three siRNAs were used for selection (Seq F, Seq I and Cy5-labeled AllStars negative control siRNA). Dissociation was performed in PBSA containing 55% mouse serum (EMD Millipore) at 37°C to mimic in vivo conditions. Unlabeled siRNA (unmodified Seq F) was added at a 100-fold molar excess over the estimated concentration of labeled siRNA. The final concentration of the competitor was between 1.7 and 2.5 μM. Six rounds of selections were performed in total, cycling between the different siRNAs twice to prevent specific binding to any one sequence. The competition time was increased from 15 minutes in the first selection cycle to 1 hour in the second cycle. Plasmids were isolated and sequenced from the enriched library as previously described (18 ).
4
Multicolor Flow Cytometry for Tregs and MDSCs
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For characterization of Tregs, PBMCs were incubated first with EMA and Gamunex™, followed by indirect staining for CD3 with a primary CD3 antibody (OKT3 supernatant) and a Pacific Orange-conjugated secondary antibody (Invitrogen). After blocking the non-specific binding of the secondary antibody with mouse serum (Merck Millipore, Darmstadt, Germany), cells were directly stained with CD4-Pacific Blue, CD45RA-Alexa Fluor-700, CD8-peridinin-chlorophyll protein (PerCP), CD279-PerCP-Cy5.5, CD127-Alexa Fluor-647 (BioLegend) and CD25-APC-Cy7 (BD Biosciences). The cells were then fixed and permeabilized using the human FoxP3 kit (BioLegend) and the cells were stained for intracellular FoxP3 using a PE-conjugated antibody (BioLegend) according to the manufacturer’s instructions.
For characterization of MDSCs, PBMCs were stained with a cocktail of lineage (Lin) markers (CD3, CD19, CD56)-Brilliant Violet 605 (BioLegend, BD Biosciences), CD14-Brilliant Violet 711 (BioLegend), CD45-V500, CD15-FITC, HLA-DR PerCP-Cy5.5, CD11b APC-Cy7, CD33 Alexa Fluor-700 (BD Biosciences), and CD124-APC (R&D Systems, Minneapolis, MN, USA). All samples were measured using a BD LSRII (BD Biosciences) immediately after staining.
For characterization of MDSCs, PBMCs were stained with a cocktail of lineage (Lin) markers (CD3, CD19, CD56)-Brilliant Violet 605 (BioLegend, BD Biosciences), CD14-Brilliant Violet 711 (BioLegend), CD45-V500, CD15-FITC, HLA-DR PerCP-Cy5.5, CD11b APC-Cy7, CD33 Alexa Fluor-700 (BD Biosciences), and CD124-APC (R&D Systems, Minneapolis, MN, USA). All samples were measured using a BD LSRII (BD Biosciences) immediately after staining.
5
Stability Evaluation of Radiolabeled Compounds
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The 177Lu-labeled compounds (~3 MBq) were incubated in PBS (300 μL) at 37 °C. The stability of the radiolabeled peptides was verified by radio-HPLC at 1, 4, and 24 h. The stability in serum was determined by incubating the radiolabeled compounds (~3 MBq) into 150 μL of mouse serum (Merck; Haarlerbergweg, The Netherlands) at 37 °C. At 1, 4, and 24 h post-incubation, the proteins were precipitated by adding an aliquot of 35 μL of the mixture to an equal volume of ACN. The vial was vortexed and centrifuged for 20 min. stability was monitored by radio-HPLC (Figures S4 and S5 ).
6
Radiolabeled DOTA-JR11 Stability Evaluation
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[225Ac]Ac-DOTA-JR11 and [177Lu]Lu-DOTA-JR11 (50 kBq and 1.2 MBq, respectively) were incubated in phosphate buffered saline (PBS; 500 and 200 µL, respectively) and mouse serum (Merck; Haarlerbergweg, The Netherlands) (250 and 100 µL, respectively) at 37 °C. The stability of [225Ac]Ac-DOTA-JR11 was verified in PBS and mouse serum at 22 h, 24 h and 27 h after incubation at 37 °C for condition 1, 2 and 3, respectively. The stability of [177Lu]Lu-DOTA-JR11 was monitored in PBS and mouse serum at 2 and 24 h post incubation. In mouse serum, the proteins were precipitated by adding an aliquot of the radiotracer to an equal volume of acetonitrile. The vial was vortexed vigorously and centrifuged for 20 min at 10,000 rpm. Stability studies were monitored by radio-HPLC (Additional file 1 : Figs. S5 and S6).
7
MSC and HGF Treatment Effects in Corneal Transplant
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For experiments investigating the effects of MSC administration, in vitro expanded and characterized MSCs were treated with either HGF‐specific small interfering RNA (siRNA) or control siRNA and injected into the tail veins of mice at 3 hours post‐transplantation (0.5 × 106 cells suspended in 100 μl of sterile saline) as previously described 11 , 14 , 23 . For experiments investigating the effects of HGF administration, 5 μl of either 0.1% murine recombinant HGF protein (R&D systems, Minneapolis, MN) in phosphate‐buffered saline (PBS) or protein control (mouse serum, Sigma‐Aldrich, St. Louis, MO) was applied topically to the ocular surface twice daily for 14 days following corneal transplantation.
8
Plasma Cholinesterase Activity Assay
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All plasma samples were measured using the Ellman assay (Ellman et al., 1961 (link)), as modified by Gard and Hooper (1993 (link)). AChE and total plasma cholinesterase (ChE) activity suppression were measured in plasma samples, with butrylcholinesterase (BChE) activity calculated as the difference between TChE and AChE. Difference from control samples indicated AChE suppression and, consequently, exposure to fenitrothion. All assay reagents were purchased from Sigma-Aldrich. Mouse serum (Sigma-Aldrich, Australia) was used as a between-assay standard and samples without the addition of enzyme were used as blanks. All samples were run at an optimal dilution ratio of 1:5 in duplicates or triplicates dependent on amount of plasma available. Detailed methodology of the assay can be found in the Buttemer et al. (2008) (link) and the appendix.
9
Serum RNA Stability Assay
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ODAGal4 (20 pmol) and double-stranded RNA oligonucleotides (5 pmol) or single-stranded RNAs (5 pmol) were mixed and then incubated at 37 °C in 10% mouse serum (Sigma-Aldrich) for various time periods. After incubation, the reaction was stopped by the addition of recombinant RNase inhibitor (10 U) (Takara Bio) and EXELDYE 6 × DNA Loading Dye (SMOBIO). In some experiments, siRNAs were digested by 7.5 µg/ml of RNase A (Thermo Fisher Scientific) in 20 mM Tris-HCl, pH 8.0, with 150 mM NaCl. The digested RNAs were then separated by a SuperSep DNA 15% native polyacrylamide gel (FUJIFILM Wako Pure Chemical) in 25 mM Tris and 192 mM glycine, stained with SYBR Green II (Takara Bio) in 0.5 × Tris borate EDTA buffer, and visualised with an LAS-4000 image analyser (FUJIFILM). The fluorescence intensity of the remaining RNAs was quantified by using ImageJ software (National Institute of Health, USA), and the half-lives of RNAs in serum were calculated by using an exponential curve-fitting algorithm in DeltaGraph (Red Rock software).
10
Immunophenotyping of Murine Immune Cells
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Cells from the peritoneum, bone marrow, and blood were incubated in PBS with 10% mouse serum (Sigma-Aldrich) for 20 minutes on ice to block nonspecific binding. Ly6G-phycoerythrin (PECy7), CD11b-PerCP Cy5.5, or Pacific Blue (BioLegend), 7/4-Alexa-647 (AbD Serotec) antimouse antibodies were added to the cell suspensions at concentrations recommend by the supplier and incubated on ice for 30 minutes in the dark. 11β-HSD1 sheep-derived antibody, generated in-house (23 (link)), was used in combination with donkey antisheep secondary antibody (Alexa Fluor 488; Invitrogen). Cells were treated with a fixation and permeabilization kit (Fix and Perm; Invitrogen) according to the manufacturer's instructions to allow for intracellular staining with the 11β-HSD1 antibody (Supplemental Figure 2 ). Blood and bone marrow cells were treated with BD lysis buffer (BD Biosciences) to eliminate red blood cells. Fluorescence was determined by FACScalibur using Cellquest (Becton Dickinson UK Ltd) or 5L LSR Fortessa using FACSDiva (Becton Dickinson UK Ltd) and analyzed using FlowJo software (Treestar).
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