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The Fluorochromasia Cell Mediated Lympholysis Assay (CML)
Published in Soldano Ferrone, B. G. Solheim, HLA Typing: Methodology and Clinical Aspects, 2019
J. W. Bruning, J. J. van der Poel, M. J. Kardol
In cellular cytotoxicity assays where effector and target cells can not be differentiated by morphology, it is usually necessary to mark the target cells for quantitation of lysis before the mixed cell incubation. The number of effector cells already dead before the test, or dying during the test, makes cytotoxicity assessment through a simple determination of the dead (or live) cell fraction impossible. To overcome this problem radioactive 51chromium (51Cr) is widely used as a viability marker of the target cells.1 We have found that carboxyfluorescein applied in the fluorochromasia method2 as described below is a good alternative.3, 4 Additional advantages aside from its non-radioactivity are the performance of the assay in Terasaki type 60-well microtest trays, in which the assay is read without sample transfer. The applied automated scanning fluorescence microscope reads one tray per minute. Further, the method requires less cells and saves disposable materials and equipment.
Designing Smart Nanotherapeutics
Published in Suresh C. Pillai, Yvonne Lang, Toxicity of Nanomaterials, 2019
A. Joseph Nathanael, Tae Hwan Oh, Vignesh Kumaravel
A supra-molecular peptide nano-sponge of (cholesterol-(K/D)n DEVDGC)3-trimaleimide units with a trigonal maleimide linker was designed for the rapid uptake by leukocytes and neural stem cells (Yapa et al. 2018). Here, ‘K’ and ‘D’ are lysine and aspartic acid. 5(6)-carboxyfluorescein was used as a model drug. The therapeutic efficacy of the nano-sponge was tested for the caspase-6 mediated release of 5(6)-carboxyfluorescein drug. The N-terminal of the peptides were capped with cholesterol and the peptides were further connected to a trimaleimide scaffold through Michael-addition (Wang et al. 2017b). It was found that the nano-sponges were efficiently taken up by the leukocytes in the peripheral blood flow. The cytotoxicity was assessed in neural progenitor cells (C17.2) by the MTT assay. The results revealed that DK20 nano-sponges are non-toxic up to 100 μM. However, a slight increase in cell proliferation is noted at low concentrations of DK20.
Interaction of The ANTI-CANDIDA Amphotericin B (And Other Polyene Antibiotics) With Lipids
Published in Rajendra Prasad, Mahmoud A. Ghannoum, Lipids of Pathogenic Fungi, 2017
Another elegant method exploits use of the fluorescence changes in the pH-sensitive fluorescent probe pyranine, encapsulated in unilamellar vesicles, to determine the permeability to saline ions.3,5,6 If, for instance, the salt is composed of K+ and an impermeant anion, an electroneutral exchange with H+ through the membrane is necessary to maintain the K+ flux. When the H+ current is facilitated by a protonophore, the drug-induced K+ flux becomes the rate-limiting step. The exchange is triggered either by a transmembrane pH gradient3 or a salt gradient.6 Another, more popular, spectrofluorimetric method involves measurement of an increase in fluorescence due to the drug-induced efflux of self-quenched encapsulated carboxyfluorescein or calcein.7 Such dyes release freely through membranes in their anionic forms and it is the efflux of their alkaline counter-ion which controls their release.8
Commercialization challenges for drug eluting contact lenses
Published in Expert Opinion on Drug Delivery, 2020
Olivia L. Lanier, Keith G. Christopher, Russell M. Macoon, Yifan Yu, Poorvajan Sekar, Anuj Chauhan
Multiple studies in the literature have incorporated liposomes into contact lens formulations. A study by Gulsen et al. [85] synthesized HEMA hydrogels with dimyristoyl phosphatidylcholine (DMPC) liposomes and showed that ophthalmic drugs were released for up to 8 days, which is significantly greater than control lenses. Another study by Danion et al. [92] immobilized PEG-biotinylated lipid liposomes to the surface of a commercial contact lens (Hioxifilcon B). First, polyethylenimine was covalently bounded onto the hydroxyl groups; then, NHS‐PEG‐biotin molecules were bound to the surface amine groups by carbodiimide chemistry. NeutrAvidin was bound to the PEG-biotin layer and the liposomes were bound to the NeutrAvidin. Consecutive layers of NeutrAvidin and liposomes were created. The lenses showed release of carboxyfluorescein for up to 12 days [92].
Optimization of an oral mucosa in vitro model based on cell line TR146
Published in Tissue Barriers, 2020
Grace C. Lin, Tamara Leitgeb, Alexandra Vladetic, Heinz-Peter Friedl, Nadine Rhodes, Angela Rossi, Eva Roblegg, Winfried Neuhaus
To examine the paracellular barrier with a second parameter, transport assays with carboxyfluorescein (Fluka, #21877) were performed. The apical media was replaced with 300 µL DMEM media or final EpiLife media (E3) containing 10 µM carboxyfluorescein. After incubation for 2 h at 37°C the apical and basolateral media was collected and the carboxyfluorescein content was given as relative fluorescence units (RFU) upon measurement at 488–520 nm with the Enspire Multimode Plate Reader (PerkinElmer). Media without carboxyfluorescein were used as negative controls and stock solutions were used to determine the permeated content of carboxyfluorescein. Through linear regression analysis, the slope of the cleared volume against time was estimated and calculated with a factor considering the growth surface area of 0.336 cm2. The permeability coefficient was calculated as the inverse of the permeability [µm/min] after subtraction of the permeability of the blank as shown in the following equation, whereby PEall refers to the overall permeability, PEblank to the permeability of the blank and PEcell gives the permeability coefficient of the cell layer as described recently.13,14
Microvesicles expressing adenosinergic ectoenzymes and their potential role in modulating bone marrow infiltration by neuroblastoma cells
Published in OncoImmunology, 2019
Fabio Morandi, Danilo Marimpietri, Alberto L. Horenstein, Maria Valeria Corrias, Fabio Malavasi
Cell proliferation was assessed using carboxyfluorescein succinimidyl ester (CFSE) dilution assay. Briefly, PB MNC from four different normal donors were stained with 1 µg/ml CFSE (Invitrogen), incubated for 15 min at 37°C, washed and then cultured in RPMI medium supplemented with 10% FBS. Cells were kept at 37°C and 5% CO2, alone or in the presence of beads coated with anti-CD3/anti-CD28 mAb (T cell activation/expansion kit, Miltenyi Biotec). Stimulated cells were cultured in 96 flat-bottom well plates (Costar Corning) in the presence or absence of MV isolated from 300 μl of BM plasma samples obtained from four different NB patients. MNC were harvested after 6 days, washed, and then stained with APC-conjugated anti-CD3 mAb (Beckman Coulter). After additional washes, cells were run on Gallios cytometer, and CFSE dilution was analyzed gating on APC+ cells, using Kaluza software (Beckman Coulter). Data were expressed as the percentage of proliferating cells.