Fluorescein Sodium

1953年，米德尔塞克斯医院的Crosland-Taylor在Nature上发表文章，将流体力学聚焦引入了细胞检测领域（这个原理，也称鞘流），是流式细胞术的基础。大约20年后，第一台商业化流式细胞仪上市。之后50年，流式细胞术被广泛应用于基础研究和临床诊断。流式细胞术的应用一.细胞表型检测免疫细胞表型是流式细胞术最突出应用。通过检测免疫细胞群的表面或细胞内标志物，对其进行鉴定和表征。流式细胞术能够精确鉴定和分类免疫细胞群，例如 T 细胞、B 细胞、NK 细胞、树突状细胞、单核细胞、巨噬细胞、血小板和粒细胞等。免疫细胞相关表型标志物T细胞标志物巨噬细胞标志物中性粒细胞标志物成纤维细胞标志物DC细胞标志物NK细胞标志物髓源抑制细胞（MDSCs）标志物B细胞标志物研究人员可以识别和量化异质群体中的各种免疫细胞亚群。临床医生可以诊断和监测各种血液系统疾病、进行免疫免疫评估（8大类免疫细胞构成与肿瘤预后）。二.细胞活力检测流式细胞术能够定量测量群体内和体外培养的活细胞和非活细胞。通过使用选择性标记活细胞或死细胞的荧光染料，流式细胞术可以提供精确可靠的活力测定，有助于确定细胞活力百分比。1980年使用H-33342检测死细胞和活细胞（Cytometry 1980）流式细胞术可以根据特定的标志物或染料区分活细胞、凋亡细胞和坏死细胞，从而更详细地了解细胞的健康和状态。通过将活力染料与细胞表面抗原、细胞内蛋白或功能测定的标记物相结合，研究人员可以在特定细胞类型或实验条件下获得有关细胞活力及其发生机制的全面信息。最常用的活性检测染料死细胞：碘化丙啶（ propidium iodide，PI）和7-AAD，与DNA结合，但只能进入膜受损的细胞，使死细胞发出荧光。凋亡细胞：annexin V：对磷脂酰丝氨酸具有强结合亲和力的蛋白质，在细胞凋亡的早期阶段暴露在质膜的外表面annexin V+PI是常用区分凋亡细胞和坏死细胞的组合。活细胞：calcein AM、CFDA（carboxyfluorescein diacetate）、FDA （fluorescein diacetate） ：进入活细胞，但只有在与细胞内酶相互作用时才会发出荧光细胞体内增殖：CFSE(CFDA-SE)穿透细胞膜，在活细胞内与胞内蛋白共价结合，水解后释放出绿色荧光。在细胞分裂增殖过程中，它的荧光强度会随着细胞的分裂而逐级递减，标记荧光可平均分配至两个子代细胞中，因此其荧光强度是亲代细胞的一半，根据这一特性，它可被用于检测细胞增殖，细胞周期的估算及细胞分裂等方面。Front Immunol. 2013 三.细胞周期分析从流式细胞术的早期开始，细胞周期分析就成为有价值的应用。原理是基于荧光和核酸的量之间的关系。常用核酸结合染料：碘化丙啶（PI），Hoechst，DAPI，7-AAD，溴化乙锭等。流式细胞术细胞周期分可以有很多方面的应用，例如，DNA/Ki67测定可以将表型选择与细胞周期分析相结合，用于监测p53细胞周期停滞，评估抗癌活，多药耐药性等。四.离子通道测定钙作为关键的第二信使，在许多细胞信号通路中起着至关重要的作用。它在免疫细胞活化中尤为重要，包括T细胞、B细胞和NK细胞。T细胞钙离子信号（Nat Rev Immunol. 2019）此外，钙信号传导还参与肥大细胞脱颗粒、神经元兴奋性、突触传递和神经递质释放至关重要。细胞脱颗粒的早期测量值是通过使用钙离子载体A23187的流式细胞术确定的。常用荧光染料：fluo-3 和indo-1。虽然Ca2+通道测量是最常见的应用之一，但其他离子如镁、钾、钠和氢也可以使用流式细胞术进行监测。五.细胞功能检测最早的检测是细胞酯酶。使用响应氧化态变化的活性染料检测粒细胞的氧化电位。例如，氢乙啶（hydroethidine）用于中性粒细胞呼吸爆发。二乙酸二氯荧光素（dichlorofluorescein diacetate），已被用于吞噬细胞功能研究。效应细胞杀伤功能，是流式细胞术的另外一个重要应用。（参考文献F., Sunga, G.M., Arango-Saenz, D., Rossetti, M. A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity. J. Vis. Exp. (126), e56191, doi:10.3791/56191 (2017).细胞因子是免疫细胞功能的重要执行分子，对科学研究，免疫细胞治疗，临床诊疗都及其关键。基于流式细胞术开发的多重细胞因子检测，已经有广泛应用。六.蛋白质工程流式细胞术和分选传统上不是蛋白质工程中最常用的技术之一。然而，近年来，在该领域的应用越来越多。Cells 2023流式细胞术被用于酶学蛋白质研究，包括细胞色素P450、葡萄糖氧化酶、几丁质酶、纤维素酶、过氧化物酶、酯酶、转移酶、β半乳糖苷酶、硫代内酯酶等。蛋白质工程，包括在基因水平上引入突变（随机或特异性），以创建由数千到数百万个单个蛋白质变体组成的文库（如上图），使用流式细胞术 每天能够分析多达 10^8–10^9 个克隆，并对具有所需特性的克隆进行分类。七.哺乳动物细胞和细菌细胞分选细胞分选是流式细胞的重要应用之一，哺乳动物细胞相对成熟，不做赘述。细菌细胞方面的应用，也逐渐开始建立。与耗时的传统琼脂铺板检测方法相比，流式分选可以快速检测和分选悬浮液中的单个细菌细胞。尽管细胞分选仪具有高性能，但它们在微生物学中的应用一直受到限制。这主要是由于微生物体积小，因此很难将它们与培养基中的细胞碎片或背景颗粒区分开来。另一个潜在的问题是，通常没有细菌菌株特有的抗体。限制细胞分选仪在细菌检测和分选中的适用性的其他因素主要与分选仪硬件功能本身有关，在流式细胞术仪器的早期，数量有限的激光器和检测器，限制一次只能使用一种或两种荧光染料。随着最新仪器的发展，多激光器和检测起的仪器被开发：包括赛默飞世尔的Bigfoot光谱细胞分选仪，BD FACSAria III分选仪，索尼MA900细胞分选仪和贝克曼库尔特的MoFlo Astrios EQ等。此外，部分致病性细菌，需要在BSL2以上的实验环境下进行，现在部分流式细胞术带有BSL 2 hood。Cells 2023八.液滴微流体液滴微流体是一个相对较新的领域，专注于皮升体积中含有细胞或DNA的离散液滴的形成，操作和分析，应用于生物学、化学、材料科学和医学。在生物学中，液滴微流体可实现单细胞分析、生物分子的高通量筛选、细胞异质性研究和药物发现。流式细胞术分析是研究单细胞的强大技术，可提供有关各种参数的宝贵信息。然而，它的测量仅限于直接连接到细胞的分子，例如表面或细胞内标记物，限制研究由细胞分泌或由DNA分子产生但不物理附的分子。液滴微流体提供了一种克服这一限制的新方法。将细胞或DNA封装在单个液滴中会产生离散的区室，从而能够分析由封装实体释放或产生的化合物。DE droplet analysis via sdDE-FACs.( Lab Chip 2020)九.下一代生物制剂生物制药已经占据了药物市场的重要份额，包括治疗性蛋白质（65%），疫苗（20%）等。通过测序（NGS）进行单B细胞库分析和克隆扩增鉴定，直接从人类幸存者克隆免疫球蛋白基因，分离出高亲和力中和抗体，加快了单克隆抗体药物的研发，然而，这种方法比较昂贵，且依旧需要后续的功能验证等。新策略可使用流式细胞术、MACS或微流体将单细胞分离与功能筛选相结合，降低开发成本并消除失败的候选药物，是流式细胞术新的应用开发方向。参考资料Crosland-Taylor, P.J. A device for counting small particles suspended in fluid through a tube. Nature 1953, 171, 37–38.Hamori, E.; Arndt-Jovin, D.J.; Grimwade, B.G.; Jovin, T.M. Selection of viable cells with known DNA content. Cytometry 1980, 1, 132–135.: Robinson, J.P.; Ostafe, R.; Iyengar, S.N.; Rajwa, B.; Fischer, R. Flow Cytometry: The Next Revolution. Cells 2023, 12, 1875. https://doi-org.libproxy1.nus.edu.sg/10.3390/ cells12141875Gennady Bocharov et al, Asymmetry of Cell Division in CFSE-Based Lymphocyte Proliferation Analysis,Front Immunol. 2013 Sep 2;4:264. doi: 10.3389/fimmu.2013.00264Brower, K.K.; Carswell-Crumpton, C.; Klemm, S.; Cruz, B.; Kim, G.; Calhoun, S.G.K.; Nichols, L.; Fordyce, P.M. Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery. Lab Chip 2020, 20, 2062–2074.本周好文推荐如需转载请联系佰傲谷并在醒目位置注明出处﹀‍‍ ·········点亮在看，传递信息♥

In the first in-human clinical trial, scientists used a novel, skull-implantable ultrasound device to open the blood-brain barrier and repeatedly permeate large, critical regions of the human brain to deliver chemotherapy that was injected intravenously. This is potentially a huge advance for glioblastoma patients because the most potent chemotherapy can't permeate the blood-brain barrier to reach the aggressive and deadly brain tumor. A major impediment to treating the deadly brain cancer glioblastoma has been that the most potent chemotherapy can't permeate the blood-brain barrier to reach the aggressive brain tumor. But now Northwestern Medicine scientists report results of the first in-human clinical trial in which they used a novel, skull-implantable ultrasound device to open the blood-brain barrier and repeatedly permeate large, critical regions of the human brain to deliver chemotherapy that was injected intravenously. The four-minute procedure to open the blood-brain barrier is performed with the patient awake, and patients go home after a few hours. The results show the treatment is safe and well tolerated, with some patients getting up to six cycles of treatment. This is the first study to successfully quantify the effect of ultrasound-based blood-brain barrier opening on the concentrations of chemotherapy in the human brain. Opening the blood-brain barrier led to an approximately four- to six-fold increase in drug concentrations in the human brain, the results showed. Scientists observed this increase with two different powerful chemotherapy drugs, paclitaxel and carboplatin. The drugs are not used to treat these patients because they do not cross blood-brain barrier in normal circumstances. In addition, this is the first study to describe how quickly the blood-brain barrier closes after sonication. Most of the blood-brain barrier restoration happens in the first 30 to 60 minutes after sonication, the scientists discovered. The findings will allow optimization of the sequence of drug delivery and ultrasound activation to maximize the drug penetration into the human brain, the authors said. "This is potentially a huge advance for glioblastoma patients," said lead investigator Dr. Adam Sonabend, an associate professor of neurological surgery at Northwestern University Feinberg School of Medicine and a Northwestern Medicine neurosurgeon. Temozolomide, the current chemotherapy used for glioblastoma, does cross the blood-brain barrier, but is a weak drug, Sonabend said. The paper will be published May 2 in The Lancet Oncology. The blood-brain barrier is a microscopic structure that shields the brain from the vast majority of circulating drugs. As a result, the repertoire of drugs that can be used to treat brain diseases is very limited. Patients with brain cancer cannot be treated with most drugs that are otherwise effective for cancer elsewhere in the body, as these do not cross the blood-brain barrier. Effective repurposing of drugs to treat brain pathology and cancer require their delivery to the brain. In the past, studies that injected paclitaxel directly into the brain of patients with these tumors observed promising signs of efficacy, but the direct injection was associated with toxicity such as brain irritation and meningitis, Sonabend said. Blood-brain barrier recloses after an hour The scientists discovered that the use of ultrasound and microbubble-based opening of the blood-brain barrier is transient, and most of the blood-brain barrier integrity is restored within one hour after this procedure in humans. "There is a critical time window after sonification when the brain is permeable to drugs circulating in the bloodstream," Sonabend said. Previous human studies showed that the blood-brain barrier is completely restored 24 hours after brain sonication, and based on some animal studies, the field assumed that the blood-brain barrier is open for the first six hours or so. The Northwestern study shows that this time window might be shorter. In another first, the study reports that using a novel skull-implantable grid of nine ultrasound emitters designed by French biotech company Carthera opens the blood-brain barrier in a volume of brain that is nine times larger than the initial device (a small single-ultrasound emitter implant). This is important because to be effective, this approach requires coverage of a large region of the brain adjacent to the cavity that remains in the brain after removal of glioblastoma tumors. Clinical trial for patients with recurrent glioblastoma The findings of the study are the basis for an ongoing phase 2 clinical trial the scientists are conducting for patients with recurrent glioblastoma. The objective of the trial -- in which participants receive a combination of paclitaxel and carboplatin delivered to their brain with the ultrasound technique -- is to investigate whether this treatment prolongs survival of these patients. A combination of these two drugs is used in other cancers, which is the basis for combining them in the phase 2 trial. In the phase 1 clinical trial reported in this paper, patients underwent surgery for resection of their tumors and implantation of the ultrasound device. They started treatment within a few weeks after the implantation. Scientists escalated the dose of paclitaxel delivered every three weeks with the accompanying ultrasound-based blood-brain barrier opening. In subsets of patients, studies were performed during surgery to investigate the effect of this ultrasound device on drug concentrations. The blood-brain barrier was visualized and mapped in the operating room using a fluorescent die called fluorescein and by MRI obtained after ultrasound therapy. "While we have focused on brain cancer (for which there are approximately 30,000 gliomas in the U.S.), this opens the door to investigate novel drug-based treatments for millions of patients who suffer from various brain diseases," Sonabend said. Other Northwestern authors include: A. Gould, C. Amidei, R. Ward, K. A. Schmidt, D.Y. Zhang, C. Gomez, J.F. Bebawy, B.P. Liu, I.B. Helenowski, R.V. Lukas, K. Dixit, P. Kumthekar, V. A. Arrieta. Lesniak, H. Zhang and R. Stupp. This work is funded by the grants 1R01CA245969-01A1 and P50CA221747 from the National Cancer Institute of the National Institutes of Health and SPORE support from the Moceri Family Foundation and the Panattoni family.