In vitro tube formation on Matrigel was analyzed, as previously described (Tang et al., 2004 (link)), with some modifications: Growth Factor Reduced Matrigel (BD Biosciences) was applied at 60 μL/well in 96-well plates and incubated at 37°C for 30 min to allow hardening. 6.0 × 103 primary lung endothelial cells medium containing 0.5% serum, were seeded on top of the Matrigel. Plates were incubated under normoxia or hypoxia (1% O2) at 37°C for 9 h. Cells were stained with Calcein AM dye (BD Bioscience) at the end of the incubation, and parameters of detected networks were analyzed using Image J software (Angiogenesis Analyzer, created by Gilles Carpentier).
Calcein am dye
Manufactured by BD
Calcein-AM dye is a fluorescent indicator used for detecting live cells. It is a cell-permeant dye that becomes fluorescent upon cleavage by intracellular esterases, indicating the presence of viable cells. The dye can be used to quantify cell viability and proliferation in various cell-based assays.
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4 protocols using calcein am dye
1
In vitro Angiogenesis Assay on Matrigel
2
Transendothelial Migration of GFP-Labeled LLC Cells
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Transendothelial migration of GFP-labeled Lewis Lung Carcinoma cells (LLCGFP) was analyzed, as previously described (Branco-Price et al., 2012 (link)) with some modifications. Endothelial cells were seeded on COSTAR transwell inserts (6.5 mm diameter, 8 μm pore; Corning, cat no. 3422) at cell density of 2.0 × 104 per well, and grown for 5 days. LLCGFP cells, pre-stained with Calcein-AM dye (BD Bioscience), were seeded on the endothelial monolayer and the inserts were incubated at 37°C for 24 h under hypoxia (1% O2), with serum-free media on both upper and lower chambers. Migrated cells were counted in five random fields per insert at 100x magnification by using the fluorescent stereomicroscope (Leica M165FC).
3
Monocyte Adhesion to HUVEC Assay
4
IgG ADCC Optimization Assay
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Example 9
Genes encoding the variable regions of a selected IgG are cloned into mammalian expression vectors encoding human Fc regions (huIgG1κ) containing amino acid mutations known to enhance Fc-receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC). Vectors are transiently transfected into HEK293/EBNA cells. After 2-7 days, IgG expression is quantified and samples of antibody are purified on protein A columns. Antibodies are then tested in ADCC assays. Neu5Gc and Neu5Ac-expressing Jurkat cell lines are used as the target cells and human peripheral blood mononuclear cells (PBMC) are used as a source of effector cells. Target cells are titrated using maximum cell lysis to determine the optimum cell density for use in multiwall plate format assay. ADCC-mutated antibody together with the non-mutated IgG are pre-incubated with target cells, effector cells are then added at varying target:effector cell ratios, and cultures are incubated at 37° C. Percentage viability is determined using Calcein-AM dye (BD Biosciences, San Jose, Calif.) release. Samples of up to 0.5 mg of ADCC-mutated IgG are subjected to further analysis.
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