Name: TCPP Bead Filtration
Uploaded: 2023-03-24T12:55:00
Description: Watch this Scientific Journal Video about TCPP Bead Filtration at JoVE.com

tcpp bead filtration

流式细胞术中的卟啉修饰微珠

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.

作者聚焦：卟啉修饰的微珠，用作流式细胞术中的补偿对照  

卟啉修饰微球用作流式细胞术补偿质控

We developed polystyrene beads modified with the porphyrin TCPP as compensation controls and multicolor flow cytometry. The beads used in this protocol have fluorescence intensity comparable to TCPP stain cells and still passed through the flow cytometer. We accomplished that and are currently working on better methods to remove the particulate byproduct.Porphyrin-modified compensation beads were preferable to TCPP stained cells as compensation controls. These speeds were stable over months without significant loss of fluorescence. Porphyrins are known to stain cancer cells selectively.The research combines the selectivity of porphyrins with quantitative flow cytometry as a powerful platform for high-throughput analysis of human samples.

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

TCPP Bead Filtration  

TCPP 微球过滤

通过用 3.20 毫升冰冷的汉克斯平衡盐溶液或 HBSS 稀释 0.8 毫升 TCPP 珠悬浮液开始 TCPP 珠过滤。以最大速度涡旋稀释的珠悬浮液15秒。然后，从一次性五毫升注射器中取出柱塞，并将注射器与直径为13毫米的五微米玻璃纤维尖端过滤器安装。接下来，向注射器中加入四毫升HBSS，然后加入0.5毫升涡旋稀释珠悬浮液。使用柱塞，以每秒约两滴的速度通过注射器过滤器过滤珠悬浮液。然后，通过过滤器将五毫升新鲜的HBSS吸入注射器中，并以每秒约两滴的速度将HBSS推入废物容器中，从而洗涤珠子。要从过滤器中取出珠子，请通过过滤器将另外五毫升新鲜的HBSS吸入注射器中。然后，小心地从注射器上取下过滤器。将珠悬浮液从注射器弹出到50毫升锥形离心管中后，将新鲜过滤器放回注射器上，重复五毫升HBSS洗涤四次。完成后，丢弃过滤器和注射器。将过滤后的珠悬浮液在室温下以1， 000g离心10分钟。从每个50毫升管中吸出上清液。轻轻重悬珠子和0.5毫升新鲜HBSS。用P1000微量移液器将组合的珠子转移到新的琥珀色玻璃小瓶中，并储存在4摄氏度。这里可以看到对含有约66%颗粒的珠悬浮液的过滤效果。过滤后，颗粒含量降至约25%，过滤的不利影响是珠子的损失和荧光强度的轻微损失。

Research

JoVE Journal

Chemistry

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 ...