Intracellular HSP72 detection in HL60 cells using a flow cytometry system based on microfluidic analysis

Gottwald, Eric; Lahni, Brigitte; Lüdke, Gerd; Preckel, Tobias; Buhlmann, Carsten

doi:10.2144/03352rr01

Cited by 11 publications

(7 citation statements)

References 16 publications

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“…Staining of intracellular HSP72 was performed according to Gottwald et al [2003]. This microfluidic Lab-on-a-chip based analysis of intracellular HSP72 was developed based on a protocol originally described by Oehler et al [2001].…”

Section: Staining Of Intracellular Hsp72 and Dna Counterstainmentioning

confidence: 99%

“…The resulting pellet was resuspended in Cell Buffer (Cell Fluorescence Assay Kit, Agilent Technologies, Waldbronn, Germany) for chip analysis. Cell fluorescence assays were performed according to Gottwald et al [2003]. The experiments were performed at least in triplicates with 3 sample preparations (control, isotype control, and heat shock treatment); 500-1000 cells per sample were analyzed.…”

Section: Staining Of Intracellular Hsp72 and Dna Counterstainmentioning

confidence: 99%

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Expression of HSP72 after ELF‐EMF exposure in three cell lines

Gottwald

Sontag

Lahni

et al. 2007

Bioelectromagnetics

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It has been reported that magnetic fields with flux densities ranging from microT to mT are able to induce heat shock factor, HSP72 mRNA or heat shock proteins in various cells. In this study we investigated changes in the HSP72 mRNA transcription level in three cell lines (HL-60, H9c2, and Girardi heart cells) and in the intracellular HSP72 protein content in two cell lines (HL-60 and Girardi heart cells) after treatment schemes using electromagnetic fields with a flux density of 2 microT to 4 mT, a frequency of 50 Hz and exposure times from 15 to 30 min. None of the treatments or modalities showed any significant effect on the HSP72 protein level, although HSP72 mRNA could be induced, at least to some extent, with one of the parameter combinations in all cell lines tested. Obviously, HSP72 mRNA transcription and translation are not necessarily coupled in certain cells. This leads to the conclusion that electromagnetic field effects on HSP72 mRNA levels are not indicative for downstream effects unless increased mRNA levels can be correlated with increased HSP72 protein levels as well.

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Section: Staining Of Intracellular Hsp72 and Dna Counterstainmentioning

confidence: 99%

Section: Staining Of Intracellular Hsp72 and Dna Counterstainmentioning

confidence: 99%

Expression of HSP72 after ELF‐EMF exposure in three cell lines

Gottwald

Sontag

Lahni

et al. 2007

Bioelectromagnetics

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“…The growing observation that small portable flow cytometers would greatly enhance the utility of flow cytometry has led to significant size and cost reductions. Approaches to reduce the cost and size of flow cytometers have included the adaptation of small low‐cost diode‐based lasers and light emitting diodes into flow cytometers (8–12), and the miniaturization of detectors and data acquisition systems in cytometers such as the Partec Model (Munster, Germany), Luminex HTS (Austin, TX), and the Microcyte Model from BioDetect (Oslo, Norway). However, all of the above‐mentioned instruments still require the use of sheath fluids to hydrodynamically focus the sample into a tight sample stream (5–10 μm in diameter) moving at a high linear velocity (5–10 m/s), through tightly focused (10–40 μm diameter) laser beams (8).…”

mentioning

confidence: 99%

Ultrasonic particle‐concentration for sheathless focusing of particles for analysis in a flow cytometer

et al. 2006

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Background: The development of inexpensive small flow cytometers is recognized as an important goal for many applications ranging from medical uses in developing countries for disease diagnosis to use as an analytical platform in support of homeland defense. Although hydrodynamic focusing is highly effective at particle positioning, the use of sheath fluid increases assay cost and reduces instrument utility for field and autonomous remote operations. Methods: This work presents the creation of a novel flow cell that uses ultrasonic acoustic energy to focus small particles to the center of a flowing stream for analysis by flow cytometry. Experiments using this flow cell are described wherein its efficacy is evaluated under flow cytometric conditions with fluorescent microspheres. Results: Preliminary laboratory experiments demonstrate acoustic focusing of flowing 10‐μm latex particles into a tight sample stream that is ∼40 μm in diameter. Prototype flow cytometer measurements using an acoustic‐focusing flow chamber demonstrated focusing of a microsphere sample to a central stream ∼40 μm in diameter, yielding a definite fluorescence peak for the microspheres as compared with a broad distribution for unfocused microspheres. Conclusions: The flow cell developed here uses acoustic focusing, which inherently concentrates the sample particles to the center of the sample stream. This method could eliminate the need for sheath fluid, and will enable increased interrogation times for enhanced sensitivity, while maintaining high particle‐analysis rates. The concentration effect will also enable the analysis of extremely dilute samples on the order of several particles per liter, at analysis rates of a few particles per second. Such features offer the possibility of a truly versatile low‐cost portable flow cytometer for field applications. © 2005 Wiley‐Liss, Inc.

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“…The need to retain the essential capabilities of existing instruments—while reducing their size, cost, and complexity—is driving the development of inexpensive flow cytometers through reductions in the size, cost, use of consumables for the fluidics, and complexity of data acquisition and optical systems. Fluidic reductions have concentrated on micro‐fluidic flow cytometry adaptations (9–12), using unfocused flow without sheath (13), or the use of alternative sample focusing forces such as acoustic (14), dielectrophoretic (15), or aerodynamic sheath (16, 17). Data systems have become ever smaller and more capable as one more benefit of the general electronics and computer revolution.…”

mentioning

confidence: 99%

“…Data systems have become ever smaller and more capable as one more benefit of the general electronics and computer revolution. Optical system reductions have used low‐power light sources such as LEDs (9, 18), the incorporation of diode pumped solid state lasers (DPSS), and red diode lasers (4, 19, 20). Although LEDs are available with impressive output intensity at very low cost, and have been demonstrated in flow cytometry (18–20), the incoherent and highly divergent light emitted makes them more difficult to incorporate into an instrument and the low effective excitation power density limits their applicability (1).…”

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confidence: 99%

Evaluation of a green laser pointer for flow cytometry

Habbersett¹,

Naivar²,

Woods³

et al. 2007

Cytometry Pt A

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Flow cytometers typically incorporate expensive lasers with high-quality (TEM00) output beam structure and very stable output power, significantly increasing system cost and power requirements. Red diode lasers minimize power consumption and cost, but limit fluorophore selection. Low-cost DPSS laser pointer modules could possibly offer increased wavelength selection but presumed emission instability has limited their use. A $160 DPSS 532 nm laser pointer module was first evaluated for noise characteristics and then used as the excitation light source in a custom-built flow cytometer for the analysis of fluorescent calibration and alignment microspheres. Eight of ten modules tested were very quiet (RMS noise 0.6% between 0 and 5 MHz). With a quiet laser pointer module as the light source in a slow-flow system, fluorescence measurements from alignment microspheres produced CVs of about 3.3%. Furthermore, the use of extended transit times and 1 mW of laser power produced both baseline resolution of all 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoerythrin molecules per particle. Data collected with the transit time reduced to 25 ls (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of $75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, and power consumption of the tested laser pointer module suggests that it may be suitable for use in conventional flow cytometry, particularly if it were coupled with cytometers that support extended transit times. Published 2007 Wiley-Liss, Inc.

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Intracellular HSP72 detection in HL60 cells using a flow cytometry system based on microfluidic analysis

Cited by 11 publications

References 16 publications

Expression of HSP72 after ELF‐EMF exposure in three cell lines

Expression of HSP72 after ELF‐EMF exposure in three cell lines

Ultrasonic particle‐concentration for sheathless focusing of particles for analysis in a flow cytometer

Evaluation of a green laser pointer for flow cytometry

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