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Measuring multiple apoptosis,,,parameters with the Agilent 2100,,,bioanalyzer

A simplified and fast protocol for the analysis of DNA fragmentation during apoptosis
This Application Note describes a simplified and fast protocol for apoptosis DNA fragmentation analysis with the Agilent 2100 bioanalyzer using the DNA LabChip kit. The combination of Annexin V, activecaspase- 3 and DNA laddering analysis on the same platform together with the Cell Fluorescence LabChip kit permits quick confirmation and quantitation of apoptosis in cell populations. A fast and simple protocol was applied for the extraction of fragmented, chromosomal DNA. The increased sensitivity and sizing accuracy of the DNA LabChip kit compared to conventional agarose gel electrophoresis also means that a significant lower amount of apoptotic cells is required for the analysis.
The Agilent 2100 bioanalyzer was introduced by Agilent Technologies as the first commerially available lab-on-a-chip analysis system for the life science laboratory using LabChip products, developed in collaboration with Caliper Technologies Corp. Chip-based approaches for a variety of separation- based techniques have been introduced, addressing DNA, RNA and protein separations.1, 2, 3. Recently the Agilent 2100 bioanalyzer has been extended to enable dual color simple flow cytometric assays4. Apoptosis assays have been demonstrated for quantitative measurements of whole cells5. The combination of cytometric and gel-like separations on the same instrument platform makes it an ideal tool for multi-parametric apoptosis quantitation and confirmation.
Apoptosis, or programmed cell death, is the outcome of a metabolic cascade that results in cells dying in a controlled manner. It is used by nature during development6, homeostasis, aging and in defense mechanisms. Apoptosis is a key factor in cancer and also suspected to be a characteristic mechanism for degenerative disorders such as Alzheimer7 or Parkinson's diseases. The process is universal and has been described in eukaryotic cells as well as in individual bacteria8, protozoa9 and amoeba10. It is a target for research scientists in a wide variety of fields. Apoptosis is characterized by a distinct set of morphological events involving plasma membrane blebbing and asymmetry loss, reduction of cell volume, loss of mitochondrial membrane potential, nuclear condensation, fragmentation of DNA at nucleosomal intervals and other cytoplasmatic changes. Ultimately fragmentation of the cell into membrane-enclosed apoptotic bodies occurs. Differential expression patterns or alternate mechanisms in the apoptosis pathways among tissues and cell lines makes it necessary to have two or even three parallel experiments to confirm, quantitate and compare kinetic events on apoptosis. The appearance of DNA laddering is unambiguously connected to apoptosis. Targeting of phosphatidyl serine (PS) in the outer leaflet of cell membrane with fluorescently labeled annexin V and antibody labeling of active-caspase- 3 in cells has been previously described in conjunction with the Agilent 2100 bioanalyzer as a quantitative measurement for apoptotic cell samples5. This Application Note describes the combined measurement of annexin V and the extracted DNA of apoptotic cells on one platform the Agilent 2100 bioanalyzer. A fast protocol for DNA preparation and recommendations for data evaluation are discussed.
Apoptosis was induced in Jurkat cells by incubation in a 5 M solution of the topoisomerase inhibitor Camptothecin (Sigma, # C9911) in medium. Cells were harvested and counted at the indicated time intervals. Cell lysis and DNA purification was performed as described in the fast protocol below. Eluted DNA was directly loaded into the chip wells, prepared according to the LabChip Reagent Kit Guide.
Fast protocol for DNA fragment purification (based on original protocol in reference 11)
Pellet 1-2 106 cells by centrifugation (2 min 400 g). Remove medium.
Add 100 L 4 C lysis solution (0. 2% Triton X, 10 mM Tris, 10 mM EDTA) and gently resuspend.
Incubate 5 min at 4 C. Centrifuge for 5 min at 13000 g.
Transfer supernatant to new vial and discard the pellet.
Purify DNA using a low volume elution method. Here a PCR purification kit* was used (QIAGEN MinElute PCR Purification Kit. Cat.28004).
Run in Agilent 2100 bioanalyzer with the DNA 12000 LabChip Kit, selecting the DNA 12000 Laddering assay from the biosizing software.

*As an alternative to the PCR purification kit, the following protocol has been successfully tested:
Add 2 L 5M NaCl to the lysate and vortex 5 seconds.
Add 200 L ice-cold absolute ethanol and vortex 5 seconds.
Incubate on ice for 10 minutes Centrifuge at 13000 g for 5 minutes. Discard supernatant.
Dry residual ethanol in speedvac or desiccator.
Resuspend precipitate in 30 L of pre-warmed (65 C) distilled water or 10 mM Tris/10 mM EDTA.
Run in Agilent 2100 bioanalyzer with the DNA 12000 LabChip kit, selecting the DNA 12000 laddering assay.
UV measurements
UV spectroscopy readings were performed to determine total DNA concentration. The Agilent 8453 UV-visible spectrophotometer was used in conjunction with a 0.2-cm light path ultra-micro cell (10 l volume). The whole spectrum (1901100 nm) was acquired and evaluation was automatically done by the Warburg-Christian method12 with internal wavelength correction at 320/10 nm.
Annexin V apoptosis measurements
A fast annexin protocol as described in reference 13 was used.
Results and Discussion
Besides DNA laddering, cytometric assays like annexin V or caspase analysis are commonly used for apoptosis detection. Figure 1A shows the analysis of annexin V in Jurkat cells on the Agilent 2100 bioanalyzer after 6 hours treatment with 5 M campthotecin. After switching the instrument to electrophoresis mode, extracted DNA from treated Jurkat cells was analyzed using the DNA 12000 LabChip kit. The characteristic DNA laddering further confirmed apoptosis in the sample (figure 1B).

Sizing information, provided by the 2100 bioanalyzer software, shows nucleosomal fragmentation of DNA by evenly distributed peaks in the electropherogram (figure 2). Apoptosis is confirmed in samples by fragmentation of the chromosomal DNA in 180200bp intervals.14

Typical DNA extraction with the described fast protocol leads to 10 L of 2-30ng DNA/L (figure 3). The DNA 12000 concentration range specification is 0.550 ng/L, so no further treatment is required. Band concentration was calculated to be approximately 1 ng/L (1 L loaded on the chip).

Even at low concentrations, apoptotic samples are clearly distinguishable from the negative control and necrotic samples (figure 4, samples 1 to 7). The positive samples in figure 4 turned out to be 35 % apoptotic as confirmed by the annexin V assay. A clear discrimination of samples that contain a lower number of apoptotic cells is achieved by overlaying negative controls and samples in the electropherogram view (figure 5).

If alternative DNA purification methods are used, special attention must be paid as the injection of large amounts of genomic DNA (like whole cell lysates) may lead to clogging of the chip. The recommended extraction protocol eliminates such interference of genomic DNA. In addition, fragmented DNA is concentrated, which permits the apoptosis measurement of low cell numbers.

A combination of complementary assays is usually required to confirm that cells are apoptotic. The 2100 bioanalyzer offers the flexibility to quantitatively measure flow cytometric parameters and subsequently perform a DNA fragmentation assay. It eliminates the need for outsourcing samples and helps keep consolidated electronic track of all results. In addition to faster results and ease-of-use, the high sensitivity and optimized protocols allow minimum cell consumption and saves sample preparation time.



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