Purpose: To study a method of the induction of dendritic cells (DCs) from rabbit peripheral blood. Methods: Peripheral
blood cells were removed from rabbit, filtered through nylon mesh. Peripheral blood mononuclear cells (PBMC) were
isolated from the blood cells by Ficoll-Hypaque centrifugation (density of 1.077g/cm3).To obtain DCs, PBMC were
cultured in RPMI1640 medium containing 10% fetal calf serum, 50U/mL penicillin and streptomycin, referred to
subsequently as complete medium, at 37°C in 5% CO2 atmosphere for 4 hours. Nonadherent cells were aspirated,
adherent cells were continued incubated in complete medium, supplemented with granulocyte/macrophage
colony-stimulating factor (GM-CSF, 50ng/ml),and interleukin 4 (IL-4, 50ng/ml) for 9 days. Fluorescein labeled
antibodies(anti-CD14, anti-HLA-DR, anti-CD86) were used to sign cells cultured for 3,6,9 days respectively, Then flow
cytometry was performed. Results: Ratio of anti-HLA-DR and anti-CD86 labeled cells increased with induction time
extension, in contrast with anti-CD14. Conclusion: Dendritic cells can be effectively induced by the method of this
experiment, cell maturation status increased with induction time extension.
It is known that singlet oxygen (1O2) is the main factor mediating cytotoxicity in photodynamic therapy (PDT). The effectiveness of a PDT treatment is directly linked to the 1O2 produced in the target. Although the luminescence from 1O2 is suggested as an indicator for evaluating photodynamic therapy, the inherent disadvantages limit its potential for in vivo applications. We have previously reported that chemiluminescence can be used to detect 1O2 production in PDT and have linked the signal to the cytotoxicity. We further our investigation for monitoring 1O2 production during PDT. The lifetime of 3,7-dihydro-6-{4-[2-(N-(5-fluoresceinyl)thioureido)ethoxy]phenyl}-2-methylimidazo {1,2-a} pyrazin-3-one-chemiluminescence (FCLA-CL) is evaluated, and the results show that it is much longer than that of direct luminescence of 1O2. A gated measurement algorithm is developed to fully utilize the longer lifetime for a clean measurement of the CL without the interference from the irradiation light. The results show that it is practically feasible to use the technique to monitor the 1O2. Compared to the direct 1O2 luminescence measurement, our new technique is sensitive and can be realized with a conventional optical detector with excellent signal-to-noise ratio. It thus provides a means for real-time in vivo monitoring of 1O2 production during PDT.
Fluoresceinyl Cypridina Lucifenn Analog (FCLA) is a reactive oxygen species (ROS) specific chemiluminescence (CL)
probe. Its detection efficiency of singlet oxygen (102)couldbe significantly enhanced in the presence of human serum
albumin (HSA). The enhancement mechanism of HSA-FCLA CL is studied in the current work by means ofdirect CL
measurement and spectroscopy. The results show that, FCLA can combine with HSA. HSA is an effective 1O2
quencher. It can react with 102 and produce a protein carbonyl group with an elevated energy state. The HSA protein
carbonyl group can transfer its energy to FCLA in the FCLA-HSA complex. Via this irradiative de-excitation pathway of
the excited FCLA, luminescence production from FCLA is greatly enhanced, in addition to the chemiluminescence from
the direct interaction of FCLA and 102 FCLA has been reported for cancer diagnosis in vivo. Considering HSA is a
natural protein that is present in all parts of a human body, the efficacy of FCLA used in vivo is expected to be enhanced
through the coupling of FCLA and HSA.
Fluoresceinyl Cypridina Luciferin Analog (FCLA) is a chemiluminescence (CL) probe for detecting reactive oxygen species (ROS). Its detection efficiency of singlet oxygen can be significantly enhanced in the presence of human serum albumin (HSA). In the current study, the mechanism of the FCLA-HSA CL system is studied by means of direct CL measurement and spectroscopy techniques. Our results show that FCLA can combine with HSA via a single binding site to form a complex. The CL efficiency of the system is largely governed by an inter-system energy transfer between the two components upon interaction with singlet oxygen. The CL production reaches maximum in a synergetic manner when equal amount of FCLA and HSA are present simultaneously, but the production is less efficient at other ratios. This suggests that the FCLA-HSA system maybe used as a singlet oxygen detecting technique with higher sensitivity compared with that of conventional CL techniques. It may also provide a potential new technique for quantitatively analyze the presence of HSA in a sample.
Photodynamic therapy (PDT) is a cancer therapy that utilizes optical energy to activate a photosensitizer drug in a target tissue. Reactive oxygen species (ROS), such as 1O2 and superoxide, are believed to be the major cytotoxic agents involved in PDT. Although current PDT dosimetry mostly involves measurements of light and photosensitizer doses delivered to a patient, the quantification of ROS production during a treatment would be the ultimate dosimetry of PDT. Technically, it is very difficult and expensive to directly measure the fluorescence from 1O2, due to its extreme short lifetime and weak signal strength. In this paper, Photofrin(R) and 635nm laser were used to generate 1O2 and superoxide in a PDT in solution. Compound 3,7- dihydro-6-{4-[2-(N’-(5-fluoresceinyl) thioureido) ethoxy] phenyl}-2- methylimidazo{1,2-a} pyrazin-3-one sodium salt,an Cyp- ridina luciferin analog commonly referred as FCLA, was used as a chemical reporter of ROS. The 532nm chemiluminescence (CL) from the reaction of the FCLA and ROS was detected with a photon multiplier tube (PMT) system operating at single photon counting mode. With the setup, we have made detections of ROS generated by PDT in real time. By varying the amount of conventional PDT dosage (photosensitizer concentration, light irradiation fluence and its delivery rate) and the amount of FCLA, the intensity of CL and its consumption rate were investigated. The results show that the intensity and temporal profile of CL are highly related to the PDT treatment parameters. This suggests that FCLA CL may provide a highly potential alternative for ROS detection during PDT.
In photodynamic therapy (PDT), a target tissue with pre-administered photosensitizer is exposed to laser light. The photochemical process produces reaction oxygen species (ROS), such as singlet oxygen and superoxide, and leads to ultimate cell death. A direct monitoring of ROS production during PDT, thus, may provide important information in both basic science and clinical practice. A cypridina luciferin analogue (FCLA) is a chemiluminescence (CL) probe that selectively detects singlet oxygen and superoxide. In this study, FCLA was used as an optical reporter of ROS produced by photosensitization reaction of Photofrin in Hanks solution and the CL was measured by a photomultiplier system operated at single photon counting mode. By varying the amount of PDT dosage (photosensitizer dose, light irradiation fluence rate) and the amount of FCLA, the intensity of CL were investigated. The results showed the FCLA concentration affects the ratio of the signal to background CL. The decay time of the photosensitized CL was approximately 10 sec., after the excitation source was turned off. In addition, the intensity of the CL-FCLA increased with increasing concentration of Photofrin and fluence rate. The work supported the potential application of FCLA-chemiluminescence probe as a dosimetric tool for PDT.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.