Journal: Molecular Imaging and Biology

Article Title: Synthesis and Optimization of the Labeling Procedure of 99m Tc-Hynic-Interleukin-2 for In vivo Imaging of Activated T lymphocytes

doi: 10.1007/s11307-009-0285-1

Figure Lengend Snippet: Analysis of 99m Tc-HYNIC-IL2 stability in human serum, saline, and DTPA solution after purification by tC2 Sep-Pak.

Article Snippet: The 99m Tc-HYNIC-IL2 complex was purified by SPE using a tC2 Sep-Pak® Cartridge (Water Corporation) using a step-gradient of H 2 O/acidified ethanol (25% phosphoric acid/ethanol 2/98), in order to remove the excess of free 99m Tc-pertechnetate and uncoupled 99m Tc-Tricine.

Techniques: Saline, Purification

Journal: Molecular Imaging and Biology

Article Title: Synthesis and Optimization of the Labeling Procedure of 99m Tc-Hynic-Interleukin-2 for In vivo Imaging of Activated T lymphocytes

doi: 10.1007/s11307-009-0285-1

Figure Lengend Snippet: SDS-PAGE of 99m Tc-IL2 performed in non-reducing conditions. Lanes 1–4 showed the unconjugated-IL2 ( lane 1 ), the HYNIC-IL2 complex after tC2 purification ( lane 2 ), radiolabeled IL2 before ( lane 3 ) and after purification with tC2 cartridge ( lane 4 ). Dimers, trimers, and 99m Tc-HYNIC-IL2 aggregates are absent.

Article Snippet: The 99m Tc-HYNIC-IL2 complex was purified by SPE using a tC2 Sep-Pak® Cartridge (Water Corporation) using a step-gradient of H 2 O/acidified ethanol (25% phosphoric acid/ethanol 2/98), in order to remove the excess of free 99m Tc-pertechnetate and uncoupled 99m Tc-Tricine.

Techniques: SDS Page, Purification

Journal: bioRxiv

Article Title: Apoptosis-mediated ADAM10 activation removes a mucin barrier promoting T cell efferocytosis

doi: 10.1101/2023.08.22.554267

Figure Lengend Snippet: a, Pre-treatment of CEM with ADAM10 inhibitor GI prior to staurosporine-induced apoptosis and mucin analysis; non-drug-treated cells = NT n = 6 - 8 independent experiments. b, ADAM10-KO (A10 KO ) CEM line analyzed for mucin expression, non-apoptotic cells (Non-apop) normalized to 100% and represented as a single bar for all groups; n = 6 independent experiments. c, Approach to detect shed CD43 ectodomain in apoptotic CEM Halo/SNAP supernatants using fluorescence correlation spectroscopy. d, Differential diffusion of soluble CD43 ectodomain in supernatants from apoptotic WT CEM (WT-apop) but not apoptotic WT CEM treated with GI (WT-apop-GI) or apoptotic ADAM10-KO (A10 KO -apop). e, Controls for CD43 ectodomain shedding. Diffusion of AF488 = free fluorochrome; Halo AF488 = fluorochrome-labelled soluble Halo tag; snCD43 HaloAF488 = free CD43 ectodomain released from CEM Halo/SNAP into supernatant; srCD43 hisAF488 = soluble recombinant A488-labeled his-tagged CD43 ectodomain. f - h, western blot of CEM cell lysates (top), β-actin loading control (middle) and supernatants (lower) from healthy and apoptotic WT or A10 KO CEM. f, CD43, g, PSGL-1, h, CD45. i - k, Densitometric quantification of western-blotted mucin bands from lysates of non-apoptotic and apoptotic WT and A10 KO CEM, band density normalized to β-actin loading control and non-apoptotic cell lysate signal intensity set to 100%; n = 2 - 5 independent experiments. i, CD43; j, PSGL-1; k, CD45. **p<0.01; ****p<0.0001; ns = not significant. Error bars represent ± 1SD.

Article Snippet: For analysis of cell phenotype during apoptosis, T cell lines, monocyte-depleted PBMCs or purified primary CD4 + T cells were centrifuged for 5 min at 400 g. Pellets were resuspended in 100 µL cold annexin V binding buffer (BD Pharmingen) for 20 min at 4°C in the dark with annexin V-FITC or - pacific blue (1:100, Biolegend), near-IR fixable viability dye (1:1000, Invitrogen) and fluorophore-conjugated primary antibodies including: anti-human CD43 sialic acid-independent clone L10 and sialic acid-dependent clone DFT-1 -APC, -PE or -AF647 (Invitrogen and Santa-Cruz) labelled at 2 µg/mL; anti-human CD45-PE (MEM-28, Abcam) at 1:100; anti-human MUC1-PE (16A, Biolegend) at 2 µg/mL; anti-human MUC24-PE (67D2, Biolegend), 0.5 µg/mL; anti-human PSGL-1-PE (TC2, Life Technologies) at 1:300; anti-human ADAM10-BV421 (11G2, BD Biosciences) at 1:100.

Techniques: Expressing, Fluorescence, Spectroscopy, Diffusion-based Assay, Recombinant, Labeling, Western Blot

Journal: bioRxiv

Article Title: Apoptosis-mediated ADAM10 activation removes a mucin barrier promoting T cell efferocytosis

doi: 10.1101/2023.08.22.554267

Figure Lengend Snippet: a - i, ImageStream TM -based analysis of apoptotic T cell uptake by MDM. a , Representative images of activated caspase-3 + (Casp3) apoptotic CEM within or adjacent to MDM following 1 h coculture, scale bar = 7 μm. b, Non-apoptotic (Non-apop) or apoptotic (Apop) WT CEM analyzed for MDM uptake after 1 or 3 h coculture by ImageStream, n = 6 independent experiments. c, Apoptotic WT, CD43 KO ( n = 7 independent experiments) or d , CD43 KO overexpressing CD43-Myc (CD43 Myc, n = 5 independent experiments) analyzed for uptake after 1 h coculture with MDM. e , Engulfed CEM are CD43 Myc -low, calculated by the ratio of % Myc + CEM in the starting culture with % Myc + CEM within MDM, n = 4 independent experiments. f, Relative MDM uptake of apoptotic WT CEM or CEM knocked out for PSGL-1 (PSGL-1 KO ) or MUC-1 (MUC1 KO ); n = 7 independent experiments. g , Relative uptake of apoptotic WT CEM, CEM knocked out for ADAM10 (A10 KO ) or A10 KO overexpressing CD43 Myc (A10 KO CD43 Myc ); n = 9 independent experiments. h , Non-apoptotic (Non-apop) or apoptotic (Apop) primary CD4 + T cells analyzed for MDM uptake after 1 or 3 h coculture as in ( b ), n = 5 - 11 independent donors. i , MDM uptake of untreated (NT), GW-treated or j , GI-treated primary T cells after 3 h coculture. k , MDM uptake of apoptotic WT or A10 KO primary T cells, n = 4 independent donors. l , Representative images of activated caspase-3 + (Casp3) apoptotic primary T cells engulfed by MDM at t = 3 h. m , Percentages of MDM with >1 engulfed primary T cell, untreated or treated with GW or GI, n = 1000 independent images analysed, MDM containing one T cell excluded to prioritize presentation of multiple uptake events. Numbers above bars represent fold-difference in T cell uptake by MDM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Error bars represent ± 1 SD.

Article Snippet: For analysis of cell phenotype during apoptosis, T cell lines, monocyte-depleted PBMCs or purified primary CD4 + T cells were centrifuged for 5 min at 400 g. Pellets were resuspended in 100 µL cold annexin V binding buffer (BD Pharmingen) for 20 min at 4°C in the dark with annexin V-FITC or - pacific blue (1:100, Biolegend), near-IR fixable viability dye (1:1000, Invitrogen) and fluorophore-conjugated primary antibodies including: anti-human CD43 sialic acid-independent clone L10 and sialic acid-dependent clone DFT-1 -APC, -PE or -AF647 (Invitrogen and Santa-Cruz) labelled at 2 µg/mL; anti-human CD45-PE (MEM-28, Abcam) at 1:100; anti-human MUC1-PE (16A, Biolegend) at 2 µg/mL; anti-human MUC24-PE (67D2, Biolegend), 0.5 µg/mL; anti-human PSGL-1-PE (TC2, Life Technologies) at 1:300; anti-human ADAM10-BV421 (11G2, BD Biosciences) at 1:100.

Techniques:

Journal: NMR in biomedicine

Article Title: Inhibition of prostate cancer proliferation by Deferiprone

doi: 10.1002/nbm.3712

Figure Lengend Snippet: Inhibition of prostate cancer cell proliferation after incubation with different concentrations of DFP. TRAMP-C2, Myc-CaP and 22rv1 cells were exposed to DFP for 48 h; 22rv1 cells were additionally exposed for 96 h because of their longer doubling times. A, Doubling times based on the number of viable cells at the time of medium change (base count, average ± SD): 48 h TRAMP-C2 group, (4.5±1.4) ×104; 48 h Myc-CaP group, (7.4±1.0) ×104; 48 h 22rv1 group, (4.8±0.8) ×104; 96 h 22rv1 group, (4.6±0.5) ×104. * mark significant differences in cell doubling time compared to untreated control cells for each cell line. B, Normalized log-lin curves (n=5 for 48 h exposure and n = 3 for 96 h exposure of 22Rv1) for each cell line, indicating the thresholds for untreated (Top), cytostatic effect (Bottom), and cytotoxic effect (inferior limit). Markers depict the experimental data, overlaid with the curve fit (solid line) and the 95% confidence bands (dotted lines). For each independent experiment, data were averaged from triplicate wells for each condition. C, IC50 and IC90 average values determined for each cell line and incubation time, from the respective log-lin curves, depicted in B.

Article Snippet: Cell migration (the wound-healing process) was monitored at different time points post-scratch (0 to 30 h for TRAMP-C2 and Myc-CaP; 0 to 192 h for 22rv1), using a Nikon Eclipse TS100 inverted microscope and an Insight Mono 2Mp microscope camera without IR filter (Spot Imaging™, Diagnostic Instruments, Sterling HTS, MI).

Techniques: Inhibition, Incubation

Journal: NMR in biomedicine

Article Title: Inhibition of prostate cancer proliferation by Deferiprone

doi: 10.1002/nbm.3712

Figure Lengend Snippet: Inhibition of cell migration due to DFP (100 μM) exposure. Scratch assays for: A, TRAMP-C2; B, Myc-CaP; C, 22rv1.

Article Snippet: Cell migration (the wound-healing process) was monitored at different time points post-scratch (0 to 30 h for TRAMP-C2 and Myc-CaP; 0 to 192 h for 22rv1), using a Nikon Eclipse TS100 inverted microscope and an Insight Mono 2Mp microscope camera without IR filter (Spot Imaging™, Diagnostic Instruments, Sterling HTS, MI).

Techniques: Inhibition, Migration

Journal: NMR in biomedicine

Article Title: Inhibition of prostate cancer proliferation by Deferiprone

doi: 10.1002/nbm.3712

Figure Lengend Snippet: Time-course effect of DFP (100 μM) on live TRAMP-C2 cell metabolism during MR perfusion experiments, as detected by 31P MRS. A-B, Representative 31P MR spectral profiles after 1 h (black) and at 32 h of cell perfusion for untreated control cells (green, A) and DFP-treated cells (red, B) – major peak assignments displayed: phosphoethanolamine (PE); phosphocholine (PC); extracellular (e) and intracellular (i) inorganic phosphate (Pi); glycerophosphoethanolamine (GPE); glycerophosphocholine (GPC); phosphocreatine (PCr); α, β and γ nucleoside-diphosphate (NDP) and -triphosphate (NTP); total nicotinamide adenine dinucleotides (NADH, NAD+, NADPH, NADP+); and diphosphodiesters (DPDE). C, Average time-course changes of Pie; and Pii, β-NTP, NADH, normalized to initial value of β-NTP (β-NTPinit), and GPC-to-PC ratio; experiments carried out in regular medium (filled circles) and in the presence of 100 μM DFP (open circles). # (black for Ctr and grey for DFP-treated cells), significantly different compared to the initial level, for each cell line (paired t-Test); * significantly different values between Ctr and DFP-treated cells at the same time point (unpaired t-Test).

Article Snippet: Cell migration (the wound-healing process) was monitored at different time points post-scratch (0 to 30 h for TRAMP-C2 and Myc-CaP; 0 to 192 h for 22rv1), using a Nikon Eclipse TS100 inverted microscope and an Insight Mono 2Mp microscope camera without IR filter (Spot Imaging™, Diagnostic Instruments, Sterling HTS, MI).

Techniques:

Journal: NMR in biomedicine

Article Title: Inhibition of prostate cancer proliferation by Deferiprone

doi: 10.1002/nbm.3712

Figure Lengend Snippet: Time-course effect of DFP (100 μM) on live TRAMP-C2 cell metabolism during perfusion experiments, as detected by 13C MRS. Position of carbon labeled by 13C from 1-13C-labeled glucose (Glc C1) depicted by number following C. A, Representative 13C-MR spectral profiles after 24 h of experiment time for untreated control (Ctr, black) and DFP-treated (DFP, red) TRAMP-C2 cells – major peak assignments displayed: glycogen (Glyc); glucose (Glc); dihydroxyacetone phosphate (DHAP); glycerol-3-phosphate (G3P); glutamate (Glu); fatty acids (FA); glutathione (GSH); pyruvate (Pyr), glutamine (Gln); alanine (Ala); and lactate (Lac). B, Average time course of 1-13C-Glc consumption and synthesis of 1-13C-Glyc, 1-13C-DHAP, 3-13C-G3P, 3-13C-Lac, 3-13C-Ala, 4-13C-Glu, and (CH2)-13C-FA, normalized to initial value of β-NTP (β-NTPinit). C, Average metabolite synthesis (consumption) rates at different time intervals. D, Biochemical model illustrating the inhibitory effect of DFP on m-Acon and the major metabolic changes detected in response to DFP exposure: increase (green); decrease (red); no change (blue). PL, phospholipids. # (black for Ctr and grey for DFP-treated cells), significantly different compared to the initial level, for each cell line (paired t-Test); * significantly different values between Ctr and DFP-treated cells at the same time point (unpaired t-Test).

Article Snippet: Cell migration (the wound-healing process) was monitored at different time points post-scratch (0 to 30 h for TRAMP-C2 and Myc-CaP; 0 to 192 h for 22rv1), using a Nikon Eclipse TS100 inverted microscope and an Insight Mono 2Mp microscope camera without IR filter (Spot Imaging™, Diagnostic Instruments, Sterling HTS, MI).

Techniques: Labeling

Journal: NMR in biomedicine

Article Title: Inhibition of prostate cancer proliferation by Deferiprone

doi: 10.1002/nbm.3712

Figure Lengend Snippet: Extracellular flux analysis in TRAMP-C2, Myc-CaP and 22rv1 cells incubated with 100 μM DFP for 24 h. A, Basal ECAR measurements for treated (DFP) and untreated (Ctr) groups, averaged over 15 min experiment time. B, Average OCR time-course measurements in TRAMP-C2 cells (single, control experiment) during sequential injections of different drugs, and ranges for calculating the functional parameter, as described by the manufacturer. C, Mitochondrial functional parameters derived from OCR time-course curves, for treated (DFP) and untreated (Ctr) groups.

Article Snippet: Cell migration (the wound-healing process) was monitored at different time points post-scratch (0 to 30 h for TRAMP-C2 and Myc-CaP; 0 to 192 h for 22rv1), using a Nikon Eclipse TS100 inverted microscope and an Insight Mono 2Mp microscope camera without IR filter (Spot Imaging™, Diagnostic Instruments, Sterling HTS, MI).

Techniques: Incubation, Functional Assay, Derivative Assay

Journal: NMR in biomedicine

Article Title: Inhibition of prostate cancer proliferation by Deferiprone

doi: 10.1002/nbm.3712

Figure Lengend Snippet: Effect of DFP (100 μM) on m-Acon expression in TRAMP-C2, Myc-CaP and 22rv1 cells after a 24 h incubation period. A, Representative Western blot membrane showing expression bands for m-Acon (top) and β-Actin (bottom) for each cell line and condition. B, Quantification of m-Acon expression by Western Blot normalized to β-Actin for each treated (DFP 100 μM) and untreated (Ctr) cell line (n=3 each). C, Effect of DFP (100 μM) on m-Acon activity in each cell line. Activity normalized to total cell protein content for each cell line. * significant difference between Ctr and DFP-treated cells for each cell line (paired t-Test).

Article Snippet: Cell migration (the wound-healing process) was monitored at different time points post-scratch (0 to 30 h for TRAMP-C2 and Myc-CaP; 0 to 192 h for 22rv1), using a Nikon Eclipse TS100 inverted microscope and an Insight Mono 2Mp microscope camera without IR filter (Spot Imaging™, Diagnostic Instruments, Sterling HTS, MI).

Techniques: Expressing, Incubation, Western Blot, Activity Assay