5 mg/ml of the anti-TM4SF5 monoclonal antibody and IgG2a control (Bethyl Laboratories) in PBS solution was adjusted to contain 50 mM borate buffer (pH 8.5). The antibodies were conjugated with DyLight 755 and purified using a DyLight 755 Antibody Labeling Kit (Thermo Scientific). Fifty micrograms of DyLight 755-labeled anti-TM4SF5 antibody or DyLight 755-labeled IgG2a control were injected into the intraperitoneal cavity of BALB/c control mice or mice bearing CT-26 cell derived tumors. The distribution of the DyLight 755-labeled antibodies was quantified by in vivo fluorescence using a real-time IVIS imaging system 200 (Xenogen Corp.). To investigate the localization of the injected DyLight 755-labeled antibodies in colon tumor tissues, the tissues were removed at 72 h. The frozen tissues were cut into 4-μm-thick slices using a cryostat and stained with SYTOX Green dye for nuclei, and the mounted samples were examined with an LSM 710.
Dylight 755 antibody labeling kit
Manufactured by Thermo Fisher Scientific
The DyLight 755 Antibody Labeling Kit is a tool used for the fluorescent labeling of antibodies. It allows for the covalent attachment of the DyLight 755 dye to the antibody, enabling its use in various fluorescence-based applications. The kit provides the necessary reagents and protocols to facilitate the labeling process.
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Lab products found in correlation
Ivis lumina xrms series 3, by PerkinElmer (2 mentions)
Living image software, by PerkinElmer (2 mentions)
Navios flow cytometer, by Beckman Coulter (1 mentions)
Kaluza software 1, by Beckman Coulter (1 mentions)
Ez link sulfo nhs lc biotinylation kit, by Thermo Fisher Scientific (1 mentions)
Streptavidin apc, by Agilent Technologies (1 mentions)
Af 647 antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using dylight 755 antibody labeling kit
1
Tracking Anti-TM4SF5 Antibody Distribution in Tumor-Bearing Mice
2
In Vivo Antibody Labeling and Imaging
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Antibodies were conjugated with DyLight 755 and purified with the DyLight 755 Antibody Labeling Kit (Thermo Fisher Scientific) in accordance with the manufacturer’s specifications (15 (link)). LoVo (2 × 106 cells per mouse) and/or Raji (107 cells per mouse) cells in 150 μl of a 1:1 mixture of PBS and Matrigel were injected subcutaneously into the thigh of BALB/c athymic nude mice. When the mean tumor volume reached approximately 200 mm3, DyLight 755–labeled antibodies (20 μg) were injected into the mice through the tail vein. Before imaging, the mice were anesthetized with 1.5 to 2.5% isoflurane (Piramal Critical Care). The whole-body distribution profiles of the antibodies were determined via in vivo fluorescence using an IVIS Lumina XRMS Series III instrument (PerkinElmer) at various time points. After the final scan, tumor tissues and normal organs were excised and imaged ex vivo. To reduce the effects of a tissue autofluorescence background, manual spectral unmixing was performed, and the fluorescence intensity of an identically sized region of interest was then quantified by radiant efficiency [photons/(s·cm2·steradian) per μW/cm2] in the Living Image Software (PerkinElmer).
3
Cell Surface Binding Assay with Phages and Antibodies
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To analyze cell surface binding, 5 x 105 cells were incubated with 1011 phages or IgG antibody at the indicated concentrations in PBS supplemented with 1% BSA and 0.1% NaN3. After 1 h at 4°C, phages were detected with anti-fd bacteriophage rabbit antibody (Sigma-Aldrich) and with donkey anti-rabbit IgG F(ab)2 fragment-FITC (Jackson Immuno Research; cat. no. 711-096-152), while IgG antibodies were detected with goat anti-human Fcγ F(ab)2 fragment–FITC (Jackson Immuno Research; cat. no. 109-096-098) or used directly labeled with DyLight755 Antibody Labeling Kit (Thermo Fisher Scientific; cat. no. 84539) according to manufacturer’s instructions. For blocking experiments, 5 µg/ml DyLight755-labeled CD38 antibodies were used after pre-incubation with 1 mg/ml unlabeled CD38 antibodies or control antibody. Samples were measured on a Navios flow cytometer and analyzed with Kaluza software 1.3 (Beckman Coulter).
4
In Vivo Antibody Biodistribution Imaging
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Antibodies were conjugated with DyLight 755 and purified using a DyLight 755 Antibody Labeling Kit (Thermo Scientific, 84538) in accordance with the manufacturer's specifications. BALB/c athymic nude mice were inoculated subcutaneously into the right thigh with SW480 (5 × 106 cells per mouse) cells in 150 μl of a 1:1 mixture of PBS/Matrigel. When the mean tumour volume reached ∼200 mm3, DyLight 755-labelled antibodies (20 μg) were intravenously injected into the mice through the tail vein. Before imaging, mice were anaesthetized with 1.5–2.5% isoflurane (Piramal Critical Care). The whole body distribution profiles of antibodies were quantified by in vivo fluorescence using the IVIS Lumina XRMS Series III (Perkin Elmer) at the indicated time intervals. After the final scan, tumour tissues (T) and normal organs (N) were excised and imaged ex vivo. To reduce the effects of tissue autofluorescence background, manual spectral unmixing was performed and the fluorescence intensity of an identically sized region of interest was then quantified by radiant efficiency (photons s−1 cm−2 steradian−1 μW−1 cm−2) using Living Image software (PerkinElmer).
5
Antibody Biodistribution in Tumor Xenografts
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Ab27, Ab27-hz9, and normal human IgG were conjugated with DyLight 755 and purified using a DyLight 755 Antibody Labeling Kit (Thermo Fisher Scientific). Seventy micrograms of dye-labeled antibody were injected into the tail vein of nude mice bearing SNU-449T7 or SNU-398 cell-derived tumors at a tumor size of ∼100 mm3. The distribution profiles of the antibody were quantified by in vivo fluorescence using an in vivo imaging system (IVIS) at 0, 24, 48, 72, and 96 h after antibody injection. The tumor was removed at 96 h after injection to determine the distribution of the DyLight755-labeled antibody in tumor tissues.
6
SARS-CoV-2 S-protein biotinylation and tetramerization
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Recombinant SARS-CoV-2 S-2P trimer was biotinylated using the EZ-Link Sulfo-NHS-LC Biotinylation Kit (ThermoFisher) and tetramerized with streptavidin-APC (Agilent) as previously described (Krishnamurty et al., 2016 (link); Taylor et al., 2012 (link)). The RBD domain of SARS-CoV-2 S was biotinylated and tetramerized with streptavidin-APC (Agilent). The APC decoy reagent was generated by conjugating SA-APC to Dylight 755 using a DyLight 755 antibody labeling kit (ThermoFisher), washing and removing unbound DyLight 755, and incubating with excess of an irrelevant biotinylated His-tagged protein. The PE decoy was generated in the same manner, by conjugating SA-PE to Alexa Fluor 647 with an AF647 antibody labeling kit (ThermoFisher).
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