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: