Six milliliters of 0.9 % NaCl solution was added to the lyophilized PyP-kit (Technescan PYP, Mallinckrodt Medical, Petten, The Netherlands). One hundred fifty microliters of the stannous ion containing PyP kit (10 times diluted) was injected through a custom-made tail vein catheter in order to reduce red blood cell-bound proteins. Fifteen minutes post-stannous PyP injection, freshly eluted pertechnetate (activity 55.5 ± 19.4 MBq, mean ± SD) was injected through the same tail vein catheter. In vivo formed 99mTc-stannous PyP binds to hydroxyapatite crystals in damaged cells, thereby acting as a molecular marker for cellular damage.
Technescan pyp
Manufactured by Mallinckrodt
Technescan PYP is a radiopharmaceutical product used in medical imaging procedures. It contains the active ingredient pyrophosphate, which is labeled with the radioactive isotope technetium-99m. The primary function of Technescan PYP is to provide a means for imaging and evaluating certain medical conditions, but a detailed description of its intended use is not available within the scope of this request.
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4 protocols using technescan pyp
1
In Vivo Radiolabeling of Cellular Damage
2
Radiolabeling of Cy5-Polymer Conjugate
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To 10 μL of Cy50.5CD10PIBMA39 (1 mg/mL in PBS), 4 μL of SnCl2.2H2O (0.44 mg/mL saline, Technescan PYP, Mallinckrodt Medical B.V.), and 100 μL of a freshly eluted 99mTc-Na-pertechnetate solution (500 MBq/mL, Mallinckrodt Medical B.V.) were added and the mixture was gently stirred in a shaking water bath for 1 h at 37 oC.19 (link) Subsequently, the labeling yield was estimated over time by ITLC analysis. Here fore 2 μL of the reaction mixture was applied on 1x7 cm ITLC-SG paper strips (Agilent Technologies, USA) for 10 min at RT with PBS as the mobile phase. After 1 h, the highest labeling yield of Cy50.5CD10PIBMA39 with 99mTc was assessed (49.6%) and the reaction mixture was purified by size exclusion chromatography with sterile PBS as the mobile phase using SephadexTM G-25 (desalting columns PD-10, GE Healthcare Europe GmbH, Freiburg, Germany). Fractions containing 99mTc- Cy50.5CD10PIBMA39 were collected and directly applied in the imaging experiments.
3
Radiolabeling and Stability of 99mTc-UBI29-41-Ad2 Conjugate
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Radiolabeling of UBI29–41-Ad2 with 99mTc (99mTc-UBI29–41-Ad2) and stability of the 99mTc-chelation
were carried out as follows: to 5 μL of UBI29–41-Cy5-Ad2 (797 μM/mL H2O), 4 μL
of SnCl2·2H2O (0.44 mg/mL saline, Technescan
PYP, Mallinckrodt Medical B.V. Petten, The Netherlands), 4 μL
of NaOH (0.1 M), and 100 μL of a freshly eluted 99mTc–Na-pertechnetate solution (1000 MBq/mL, Mallinckrodt Medical
B.V.) were added, and the mixture was gently stirred in a shaking
water bath for 1 h at 37 °C.15 (link),18 (link) The schematic
structure of 99mTc-UBI29–41-Ad2 is depicted inFigure S3 . The stability
of the radiolabeling was determined at 37 °C at various intervals
up to 24 h in 1 mL of PBS or fetal calf serum (20% vol/vol, FCS, Life
Technologies Inc. CA). The release of radioactivity was assessed by
instant thin-layer chromatography (ITLC) on 1 × 7 cm ITLC-SG
paper strips (Agilent Technologies, USA) using PBS as the mobile phase
(Figure S4A ).
were carried out as follows: to 5 μL of UBI29–41-Cy5-Ad2 (797 μM/mL H2O), 4 μL
of SnCl2·2H2O (0.44 mg/mL saline, Technescan
PYP, Mallinckrodt Medical B.V. Petten, The Netherlands), 4 μL
of NaOH (0.1 M), and 100 μL of a freshly eluted 99mTc–Na-pertechnetate solution (1000 MBq/mL, Mallinckrodt Medical
B.V.) were added, and the mixture was gently stirred in a shaking
water bath for 1 h at 37 °C.15 (link),18 (link) The schematic
structure of 99mTc-UBI29–41-Ad2 is depicted in
of the radiolabeling was determined at 37 °C at various intervals
up to 24 h in 1 mL of PBS or fetal calf serum (20% vol/vol, FCS, Life
Technologies Inc. CA). The release of radioactivity was assessed by
instant thin-layer chromatography (ITLC) on 1 × 7 cm ITLC-SG
paper strips (Agilent Technologies, USA) using PBS as the mobile phase
(
4
Radiolabeling of UBI29-41-Cy5-N3 with 99mTc
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Radiolabeling of UBI29–41-Cy5-N3 with 99mTc was performed according
to a previous procedure.32 (link) In short, to
5 μL of UBI29–41-Cy5-N3 (15 μM
in PBS), 4 μL of SnCl2·2H2O (0.44
mg/mL saline, Technescan PYP, Mallinckrodt Medical B.V. Petten), 4
μL of NaOH (0.1 M), and 100 μL of a freshly eluted 99mTc-Na-pertechnetate solution (1000 MBq/mL, Mallinckrodt
Medical B.V.) were added, and the mixture was gently stirred in a
shaking water bath for 1 h at 37 °C.32 (link) After 1 h, the reaction mixture was purified by size-exclusion chromatography
with sterile PBS as the mobile phase using Sephadex G-25 desalting
columns (PD-10, GE Healthcare Europe GmbH). Fractions containing 99mTc-UBI29–41-Cy5-N3, counted
for radioactivity in a dose-calibrator, were collected and directly
used for labeling of bacteria. The stability of 99mTc-labeling
was determined after incubation of 10 μL of tracer at rt in
1 mL of PBS or fetal-calf serum (20%, v/v, FCS, Life Technologies
Inc.), and samples were analyzed at various intervals until 24 h.
The release of radioactivity was determined by instant thin-layer
chromatography (ITLC) on 1 × 7 cm ITLC-SG paper strips (Agilent
Technologies) with PBS as the mobile phase. A schematic depiction
of 99mTc-UBI29–41-Cy5-N3 is
shown inFigure S7 .
to a previous procedure.32 (link) In short, to
5 μL of UBI29–41-Cy5-N3 (15 μM
in PBS), 4 μL of SnCl2·2H2O (0.44
mg/mL saline, Technescan PYP, Mallinckrodt Medical B.V. Petten), 4
μL of NaOH (0.1 M), and 100 μL of a freshly eluted 99mTc-Na-pertechnetate solution (1000 MBq/mL, Mallinckrodt
Medical B.V.) were added, and the mixture was gently stirred in a
shaking water bath for 1 h at 37 °C.32 (link) After 1 h, the reaction mixture was purified by size-exclusion chromatography
with sterile PBS as the mobile phase using Sephadex G-25 desalting
columns (PD-10, GE Healthcare Europe GmbH). Fractions containing 99mTc-UBI29–41-Cy5-N3, counted
for radioactivity in a dose-calibrator, were collected and directly
used for labeling of bacteria. The stability of 99mTc-labeling
was determined after incubation of 10 μL of tracer at rt in
1 mL of PBS or fetal-calf serum (20%, v/v, FCS, Life Technologies
Inc.), and samples were analyzed at various intervals until 24 h.
The release of radioactivity was determined by instant thin-layer
chromatography (ITLC) on 1 × 7 cm ITLC-SG paper strips (Agilent
Technologies) with PBS as the mobile phase. A schematic depiction
of 99mTc-UBI29–41-Cy5-N3 is
shown in
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