Violet laser diodes as light sources for cytometry

Shapiro, Howard M.; Perlmutter, Nancy G.

doi:10.1002/1097-0320(20010601)44:2<133::aid-cyto1092>3.0.co;2-s

Cited by 58 publications

(38 citation statements)

References 9 publications

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“…Briefly, cells were washed once in phenolfree Hanks' balanced salt solution (HBSS) supplemented with 2% FCS (HBSS/2) and then resuspended with HBSS/2 containing 10 mol Hoechst dye (Hst) (Molecular Probes, Eugene, OR). Hoechst 33342 was used for sorting with a UV laser (13), and Hoechst 34580 was used for sorting with a violet laser (28). The spleen cells were incubated for 45 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Role of B-Cell Proliferation in the Establishment of Gammaherpesvirus Latency

Moser

Upton

Allen

et al. 2005

J Virol

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Murine gammaherpesvirus 68 (␥HV68) provides a tractable small animal model with which to study the mechanisms involved in the establishment and maintenance of latency by gammaherpesviruses. Similar to the human gammaherpesvirus Epstein-Barr virus (EBV), ␥HV68 establishes and maintains latency in the memory B-cell compartment following intranasal infection. Here we have sought to determine whether, like EBV infection, ␥HV68 infection in vivo is associated with B-cell proliferation during the establishment of chronic infection. We show that ␥HV68 infection leads to significant splenic B-cell proliferation as late as day 42 postinfection. Notably, ␥HV68 latency was found predominantly in the proliferating B-cell population in the spleen on both days 16 and 42 postinfection. Furthermore, virus reactivation upon ex vivo culture was heavily biased toward the proliferating B-cell population. DNA methyltransferase 1 (Dnmt1) is a critical maintenance methyltransferase which, during DNA replication, maintains the DNA methylation patterns of the cellular genome, a process that is essential for the survival of proliferating cells. To assess whether the establishment of ␥HV68 latency requires B-cell proliferation, we characterized infections of conditional Dnmt1 knockout mice by utilizing a recombinant ␥HV68 that expresses Cre-recombinase (␥HV68-Cre). In C57BL/6 mice, the ␥HV68-Cre virus exhibited normal acute virus replication in the lungs as well as normal establishment and reactivation from latency. Furthermore, the ␥HV68-Cre virus also replicated normally during the acute phase of infection in the lungs of Dnmt1 conditional mice. However, deletion of the Dnmt1 alleles from ␥HV68-infected cells in vivo led to a severe ablation of viral latency, as assessed on both days 16 and 42 postinfection. Thus, the studies provide direct evidence that the proliferation of latently infected B cells is critical for the establishment of chronic ␥HV68 infection.Epstein-Barr virus (EBV) is a human lymphotropic virus that establishes a life-long infection, predominantly in memory B cells (17,35,36). EBV has been associated with numerous human malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, and posttransplantation lymphoproliferative disease, and is capable of transforming B cells in vitro (34). Since EBV has a very limited host range, the study of this virus in vivo is difficult. Murine gammaherpesvirus 68 (␥HV68) is a closely related gammaherpesvirus that infects small rodents and has recently been used to characterize the pathogenesis of gammaherpesviruses in vivo (31). Similar to EBV, ␥HV68 establishes latency in B cells and persists for the lifetime of the host in the memory B-cell compartment (10, 42).In the peripheral blood, EBV latency is restricted to resting memory B cells, while at sites of active virus replication, such as the tonsils, EBV infection of naïve, germinal-center, and memory B cells can be detected (3, 4). The current model for the establishment of EBV latency suggests that virus infection initiates t...

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Section: Methodsmentioning

confidence: 99%

Role of B-Cell Proliferation in the Establishment of Gammaherpesvirus Latency

Moser

Upton

Allen

et al. 2005

J Virol

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“…In addition to the traditional blue-green 488 nm laser wavelength red lasers, usually red laser diodes in the 635-645 nm range and violet laser diodes, which emit between 400 and 410 nm, are the most frequent additions (2)(3)(4). Violet laser diodes have been particularly useful for increasing the number of fluorescent probes that can be used simultaneously (5).…”

mentioning

confidence: 99%

Near‐ultraviolet laser diodes for brilliant ultraviolet fluorophore excitation

Telford

2015

Cytometry Pt A

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Although multiple lasers are now standard equipment on most modern flow cytometers, ultraviolet (UV) lasers (325-365 nm) remain an uncommon excitation source for cytometry. Nd:YVO 4 frequency-tripled diode pumped solid-state lasers emitting at 355 nm are now the primary means of providing UV excitation on multilaser flow cytometers. Although a number of UV excited fluorochromes are available for flow cytometry, the cost of solid-state UV lasers remains prohibitively high, limiting their use to all but the most sophisticated multilaser instruments. The recent introduction of the brilliant ultraviolet (BUV) series of fluorochromes for cell surface marker detection and their importance in increasing the number of simultaneous parameters for high-dimensional analysis has increased the urgency of including UV sources in cytometer designs; however, these lasers remain expensive. Near-UV laser diodes (NUVLDs), a direct diode laser source emitting in the 370-380 nm range, have been previously validated for flow cytometric analysis of most UV-excited probes, including quantum nanocrystals, the Hoechst dyes, and 4 0 ,6-diamidino-2-phenylindole. However, they remain a little-used laser source for cytometry, despite their significantly lower cost. In this study, the ability of NUVLDs to excite the BUV dyes was assessed, along with their compatibility with simultaneous brilliant violet (BV) labeling. A NUVLD emitting at 375 nm was found to excite most of the available BUV dyes at least as well as a UV 355 nm source. This slightly longer wavelength did produce some unwanted excitation of BV dyes, but at sufficiently low levels to require minimal additional compensation. NUVLDs are compact, relatively inexpensive lasers that have higher power levels than the newest generation of small 355 nm lasers. They can, therefore, make a useful, cost-effective substitute for traditional UV lasers in multicolor analysis involving the BUV and BV dyes.

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“…These lasers are small and durable enough for integration into small benchtop cuvette-based instrumentation, and often possess sufficient power levels to accommodate stream-in-air cell sorting (2). Ultraviolet (370-380 nm), violet (395-415 nm), and blue (435-445 nm) laser diodes have all been incorporated into flow cytometers and are being used for exciting a wide range of useful fluorescent probes (3)(4)(5)(6). DPSS lasers are now available in the blue-green range (473-515 nm), with the DPSS 488 nm variant replacing traditional ion lasers for many instrument applications.…”

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confidence: 99%

Solid state yellow and orange lasers for flow cytometry

et al. 2008

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Diode and DPSS lasers emitting a variety of wavelengths are now commonly incorporated into flow cytometers, greatly increasing our capacity to excite a wide variety of fluorochromes. Until recently, however, virtually no practical technology existed for generating yellow or orange laser light for flow cytometry that was compatible with smaller instrumentation. In this study, we evaluate several new solid state laser systems that emit from the 570 to 600 nm as excitation sources for flow cytometry. DPSS 580, 589, and 592 nm sources were integrated into a cuvette-based flow cytometer (BD LSR II) and a stream-in-air cell sorter (FACSVantage DiVa), and used to excite a variety of yellow, orange, and red excited fluorochromes, including Texas Red, APC, and its tandem conjugates, and the genetically encoded red fluorescent protein HcRed and the more recently developed Katushka. All laser sources were successfully incorporated into the indicated flow cytometry platforms. The yellow and orange sources (particularly 592 nm) were ideal for exciting Texas Red, and provided excitation of APC and its tandems that was comparable to a traditional red laser source, albeit at higher power levels than red sources. Yellow and orange laser light was optimal for exciting HcRed and Katushka. Practical yellow and orange laser sources are now available for flow cytometry. This technology fills an important gap in the laser wavelengths available for flow, now almost any fluorochrome requiring visible light excitation can be accommodated. recently produced a variety of novel laser sources that have proven useful for flow and image cytometry (1). These lasers are small and durable enough for integration into small benchtop cuvette-based instrumentation, and often possess sufficient power levels to accommodate stream-in-air cell sorting (2). Ultraviolet (370-380 nm), violet (395-415 nm), and blue (435-445 nm) laser diodes have all been incorporated into flow cytometers and are being used for exciting a wide range of useful fluorescent probes (3-6). DPSS lasers are now available in the blue-green range (473-515 nm), with the DPSS 488 nm variant replacing traditional ion lasers for many instrument applications. Green (532 nm) and green-yellow (561 nm) DPSS lasers have proven useful for efficient excitation of phycoerythrin and its tandems, as well as rhodamine-based fluorochromes and GFP-like long-wavelength fluorescent proteins (2,7,8). These lasers are also starting to appear on commercial flow cytometry instrumentation, vastly improving our ability to utilize the full spectrum of fluorescent probes available for biomedical analysis.However, until very recently, a significant gap existed in the range of laser wavelengths available for flow cytometry. The yellow to orange bandwidth ($570-620 nm) was very difficult to achieve using existing technology. Yellow and orange

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Violet laser diodes as light sources for cytometry

Cited by 58 publications

References 9 publications

Role of B-Cell Proliferation in the Establishment of Gammaherpesvirus Latency

Role of B-Cell Proliferation in the Establishment of Gammaherpesvirus Latency

Near‐ultraviolet laser diodes for brilliant ultraviolet fluorophore excitation

Solid state yellow and orange lasers for flow cytometry

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