tcpp bead filtration

Perlas modificadas con porfirina en citometría de flujo

Porphyrins have been of interest for many years in the biomedical field owing to their fluorescence and tumor-targeting properties1,2,3. Therapeutic applications such as photodynamic therapy (PDT) and sonodynamic therapy (SDT) entail the systemic administration of a porphyrin to a cancer patient, the accumulation of the drug in the tumor, and the localized exposure of the tumor to a laser light of a specific wavelength or ultrasound. The exposure to laser light or ultrasound leads to the generation of reactive oxygen species by the porphyrin and subsequent cell death4,5. In photodynamic diagnosis (PDD), porphyrin fluorescence is used to distinguish cancer cells from normal cells6. In this context, protoporphyrin IX, a natural fluorescent porphyrin that accumulates in tumors upon the systemic or local injection of its precursor, 5-aminolevulinic acid (5-ALA), is used to identify gastrointestinal stromal tumors, bladder cancer, and brain cancer7,8. More recently, 5-ALA treatment was explored as an approach to detect minimal residual disease in multiple myeloma9. Our laboratory has been using the tetraaryl porphyrin TCPP (5,10,15,20-tetrakis-(4-carboxyphenyl)-21,23H-porphine) for its ability to selectively stain lung cancer cells and cancer-associated cells in human sputum samples, which is a property that has been exploited in slide-based and flow cytometric diagnostic assays10.
Some porphyrins are bifunctional in that they can be used as therapeutic and diagnostic agents2,11. In biomedical research, such bifunctional porphyrins are used to evaluate how their ability to selectively target and kill cancer cells is a function of their structure as well as how it is affected by the presence of other compounds12,13,14,15,16. Both the cellular uptake of porphyrins and their cytotoxicity can be measured on a flow cytometric platform in a high-throughput manner. The absorption and emission spectra of fluorescent porphyrins are complex, but most flow cytometric platforms are equipped to correctly identify them. The absorption spectrum of fluorescent porphyrins is characterized by a strong absorption band in the 380-500 nm range, known as the Soret band. Two to four weaker absorption bands are generally observed in the 500-750 nm range (Q bands)17. A blue&#160;488 nm laser, present in most flow cytometers, or a violet laser (405 nm) can generate light of the appropriate wavelength to excite porphyrins. The emission spectra of porphyrins typically display peaks in the 600-800 nm range18, which results in very little spectral overlap with fluorescein isothiocyanate or phycoerythrin (PE) fluorophores but considerable overlap with other often-used fluorophores, such as allophycocyanin (APC), as well as tandem fluorophores, such as PE-Cy5 and others. Therefore, when using porphyrins in multi-color flow cytometry assays, single-fluorophore controls are essential to adequately correct the spillover of fluorescence in channels other than the one designated to measure the porphyrin&#39;s fluorescence.
Ideally, the single-fluorophore controls used to calculate the spillover matrix for a panel of fluorophores (also called &#34;compensation controls&#34;) should consist of the same cell type(s) as the sample. However, using the sample for this purpose is not optimal if there is very little sample to begin with or if the target population within the sample is very small&#160;(for example, if one wants to look at minimal residual disease or cancer cells at the early stages of the disease). A useful alternative to cells is beads coupled with the same fluorophore that is used to analyze the sample. Many such beads are commercially available; these beads are either prelabeled with the desired fluorophore (prelabeled fluorophore-specific beads)19,20, or a fluorescently labeled antibody can be attached to them (antibody capture beads)20,21. While commercial compensation beads are available for many fluorophores, such beads are unavailable for porphyrins, despite their increasing use in basic and clinical research.
In addition to sample preservation and appropriately sized positive versus negative populations, the other advantages of using beads as compensation controls are the ease of preparation, low background fluorescence, and excellent stability over time22. The potential disadvantage of using beads as a compensation control is that the emission spectrum of the fluorescent antibody captured on beads may differ from that of the same antibody used to label the cells. This may be of specific importance when using a spectral flow cytometer20. Therefore, the development of beads as a compensation control needs to be performed on the flow cytometer that will be used for the assay for which the beads are developed. Moreover, the development of the beads needs to include a comparison with cells labeled with the same fluorescent staining reagent.
Here, we describe the preparation of TCPP amine-functionalized polystyrene compensation beads, whose median fluorescence intensity in the detection channel was comparable to that of TCPP-labeled cells in sputum, and their use as compensation controls for flow cytometry. The autofluorescence of equivalent, non-functionalized beads was sufficiently low for their use as negative fluorescence compensation controls. In addition, these beads demonstrated stability in storage for nearly 1 year.

Autor destacado: Perlas modificadas con porfirina para su uso como controles de compensación en citometría de flujo  

Perlas modificadas con porfirina para uso como controles de compensación en citometría de flujo

Watch this Scientific Journal Video about TCPP Bead Filtration at JoVE.com

TCPP Bead Filtration  

Filtración de cuentas TCPP

Comience la filtración de perlas TCPP diluyendo 0.8 mililitros de la suspensión de perlas TCPP con 3.20 mililitros de solución salina balanceada de Hanks helada, o HBSS. Vortex la suspensión de cuentas diluidas a velocidad máxima durante 15 segundos. Luego, retire el émbolo de una jeringa desechable de cinco mililitros y coloque la jeringa con un filtro de punta de fibra de vidrio de cinco micrómetros con un diámetro de 13 milímetros.A continuación, agregue cuatro mililitros de HBSS a la jeringa antes de agregar 0,5 mililitros de la suspensión de perlas diluidas de vórtice. Usando el émbolo, filtre la suspensión de perlas a través del filtro de la jeringa a aproximadamente dos gotas por segundo. Luego, lave las perlas extrayendo cinco mililitros de HBSS fresco en la jeringa a través del filtro y empujando el HBSS hacia el contenedor de desechos a aproximadamente dos gotas por segundo.Para retirar las perlas del filtro, extraiga otros cinco mililitros de HBSS fresco en la jeringa a través del filtro. Luego, retire el filtro con cuidado de la jeringa. Después de expulsar la suspensión del talón de la jeringa a un tubo de centrífuga cónica de 50 mililitros, vuelva a colocar el filtro nuevo en la jeringa y repita el lavado HBSS de cinco mililitros cuatro veces.Una vez hecho esto, deseche el filtro y la jeringa. Centrifugar las suspensiones de perlas filtradas a temperatura ambiente durante 10 minutos a 1.000 g. Aspire el sobrenadante de cada tubo de 50 mililitros.Vuelva a suspender suavemente las perlas y 0,5 mililitros de HBSS fresco. Transfiera las perlas combinadas con una micropipeta P1000 a un nuevo vial de vidrio de color ámbar y guárdelas a cuatro grados centígrados. El efecto de filtración en una suspensión de perlas que comprende aproximadamente 66% de partículas se ve aquí.Después de la filtración, el contenido de partículas disminuyó a aproximadamente 25%Los efectos adversos de la filtración son la pérdida de perlas y una ligera pérdida en la intensidad de fluorescencia.

Research

JoVE Journal

Chemistry

TCPP Bead Filtration

150 vistas. Comience la filtración de perlas TCPP diluyendo 0.8 mililitros de la suspensión de perlas TCPP con 3.20 mililitros de solución salina balanceada de Hanks helada, o HBSS. Vortex la suspensión de cuentas diluidas a velocidad máxima durante 15 segundos. Luego, retire el émbolo de una jeringa desechable de cinco mililitros y coloque la jeringa con un filtro de punta de fibra de vidrio de cinco micrómetros con un diámetro de 13 milímetros.A continuación, agregue cuatro mililitros ...

Video: TCPP Bead Filtration  

Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or more fluorescent reagents, each functionalized with a different fluorescent molecule (fluorophore) that binds to cells selectively based on their phenotypic characteristics, such as cell surface antigen expression. The intensity of fluorescence from each reagent bound to cells can be measured on the flow cytometer using channels that detect a specified range of wavelengths. When multiple fluorophores are used, the light from individual fluorophores often spills over into undesired detection channels, which requires a correction to the fluorescence intensity data in a process called compensation. 
Compensation control particles, typically polymer beads bound to a single fluorophore, are needed for each fluorophore used in a cell labeling experiment. Data from compensation particles from the flow cytometer are used to apply a correction to the fluorescence intensity measurements. This protocol describes the preparation and purification of polystyrene compensation beads covalently functionalized with the fluorescent reagent meso-tetra(4-carboxyphenyl) porphine (TCPP) and their application in flow cytometry compensation. In this work, amine-functionalized polystyrene beads were treated with TCPP and the amide coupling reagent EDC (N-(3-dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride) at pH 6 and at room temperature for 16 h with agitation. The TCPP beads were isolated by centrifugation and resuspended in a pH 7 buffer for storage. TCPP-related particulates were observed as a byproduct. The number of these particulates could be reduced using an optional filtration protocol. The resultant TCPP beads were successfully used on a flow cytometer for compensation in experiments with human sputum cells labeled with multiple fluorophores. The TCPP beads proved stable following storage in a refrigerator for 300 days.

The protocol describes how porphyrin-based compensation beads for flow cytometry are prepared by the reaction of amine-functionalized polystyrene beads with the porphyrin TCPP and the amide coupling reagent EDC. A filtration procedure is used to reduce the particulate byproducts.

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or ...