Nexcelom Bioscience
Cell Viability and Necrosis Cellometer Applications

Detection of Cell Viability and Necrosis

Compatible with Cellometer:

Overview of Viability and Necrosis

Measuring viability and necrosis are an important component of pharmaceutical and academic research. Identifying viable cells in harvested primary samples and in tissue cultured cells provides researchers the ability to determine the condition of their samples.  Furthermore, the ability to quickly determine the cytotoxic effect of chemical compounds on cancer cells allows researchers to efficiently identify potential drug candidates for further development in the pharmaceutical discovery pipeline.

Necrosis is most often characterized by irreversible cellular damage which includes but not limited to: cytoplasmic swelling, permanent cell membrane damage, organelle breakdown, and eventually the release of cellular contents into the surrounding medium.

In non-viable cells the loss of cellular membrane integrity allows for membrane exclusion dyes such as propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green/red, and others to freely diffuse into the cell and bind to its DNA. Because these dyes only enter cells with compromised cell membranes, early apoptotic and healthy cells will not be stained, while dead or dying cells will be stained. The emitted fluorescent signal is captured by the Cellometer and an image is generated. Both brightfield and fluorescent images are captured and enumerated by the Cellometer.

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Sample Detection Using Cellometer

Detection of Cell Viability and Necrosis Using Cellometer Vision

With the Cellometer Vision, just 20 µl of sample is added to the Cellometer Counting Chamber. Imaging and analysis of the samples is completed in less than 30 seconds. Bright field and fluorescent cell images can be viewed to check cell morphology and verify cell counting. Total cell count, concentration, and mean diameter are automatically displayed.

Pipette 20 µl of cell sample into a disposable counting slide

1. Pipette 20 µl of cell sample into a disposable counting slide.

Insert slide in to cell counter

2. Insert slide into the instrument

select assay

3. Select assay from a drop down menu

cell count, concentration, diameter, and percent viability

4. Click count, acquire image and view cell count, concentration, diameter

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Detection of Cell Viability and Necrosis Using Cellometer Auto 2000

Simple, User-friendly Procedure

1. Pipette 20 µl

2. Insert Slide

3. Select Assay & Click Count

4. Results in 30 seconds!

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Temperature Induced Necrosis

Jurkat cells were exposed to various temperatures for 20 minutes and stained with propidium iodide. At 37 °C, the standard incubation temperature, cells remained healthy and very few necrotic cells were detected. The viability of the sample was determined to be 93.8 %. As the temperature was increased the percent of PI positive cells increased from 6.2% at 37 °C to 89.8% at 60 °C.

Temperature induced necrosis
 
37°C
45°C
50°C
55°C
60°C
Bright Field
1237
1312
1099
1410
1097
PI Positive
79
169
176
623
983
Viability
93.8%
87.2%
84.0%
55.8%
10.2%

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Examples of Cell Viability Measurement Using PI

Below are images of cultured cells that have been stained with PI and analyzed using a Vision instrument. For each example a bright field and a fluorescent image is provided. In bright-field, live cells appear round with well-defined membranes and bright centers (example cells circled in blue). The cell morphology of dead cells is often distinctly different from live cells. Non-viable cells often have poorly defined faint cell membranes and centers (example cells circled in red). Only the non-viable cells are stained with PI and are seen in the fluorescent images.

3T3 Cells

3T3 Cells

HEK293 Cells

HEK293 Cells

K562 Cells

K562 Cells

PC3 Cells

PC3 Cells

CHO Cells

CHO Cells

Hi5 Cells

Hi5 Cells

LnCap Cells

LnCap Cells

Jurkat Cells

Jurkat Cells

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Viability Measurement in Primary Clinical Cells

Detection of viable cells in primary clinical samples is an important step in evaluating the sample quality and percent of viable cells.

Tumor Digest

tumor digest cells
Learn more about Immunology »

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Viability Measurement in PBMCs

The quality of peripheral blood mononuclear cell (PBMC) samples after isolation, during experimentation, as well as before and after cryopreservation needs to be assessed. The two micrographs (below) show live mononuclear cells stained with acridine orange in green, and dead cells stained with PI in red.

PBMC

PBMC - peripheral blood mononuclear cell
Learn more about PBMC »

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Viability Measurement in GFP Expressing Cells

By staining the cells with propidium iodide, in a single assay, we can monitor the quality of the cell culture during GFP expression. In this example, healthy mouse embryonic stem cells that are expressing GFP are shown to be negative for propidium iodide staining, while other cells in the culture are PI positive.

GFP

GFP expressing cells
Learn more about GFP »

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Other Membrane Exclusion Dyes

Other than propidium iodide, other fluorescent membrane exclusion dyes can also be used to measure cell viability.

Jurkat Cells – 7AAD

Jurkat Cells – 7AAD

Bone Marrow – Ethidium Bromide

Bone Marrow – Ethidium Bromide

PC3 Cells – DRAQ7

PC3 Cells – DRAQ7

CHO Cells – SYTOX Green

CHO Cells – SYTOX Green

Jurkat Cells – SYTOX Red

Jurkat Cells – SYTOX Red

Jurkat Cells – DAPI

Jurkat Cells DAPI

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Conclusion and Cellometer Selection

Performing cell viability assays can help researchers determine the health of their cultures during experimentation. Here we have used multiple fluorescent membrane exclusion dyes on a variety of primary and cultured samples to successfully demonstrate the versatility of the Cellometer system to accurately measure cellular viability. This platform can also be used as a tool to detect necrotic cells.

Please contact an application specialist to determine which Cellometer is right for you!

Ask an Applications Specialist

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Publications Using Cellometer for Cell Viability and Necrosis Measurement

  • Yedjou C, Izevbigie E, Tchounwou P. (2013) Vernonia amygdalina—Induced Growth Arrest and Apoptosis of Breast Cancer (MCF-7) Cells. Pharmacology & Pharmacy 4 (1): 93-99
  • Chan LL, Wilkinson AR, Paradis BD, Lai N. (2012) Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods. Journal of Fluorescence 22(5):1301-1311
  • Holmuhamedov EL, Czerny C, Beeson CC, et al. Ethanol suppresses ureagenesis in rat hepatocytes: role of acetaldehyde. (2012) Journal of Biological Chemistry 287(10):7692-7700
  • Ranguelova K, Rice AB, Khajo A, et al. Mason RP. (2012) Formation of reactive sulfite-derived free radicals by the activation of human neutrophils: An ESR study. Free Radical Biology & Medicine 52:1264-1271
  • Lugli E, Gattinoni L, Roberto A, et al. (2012) Identification, isolation and invitro expansion of human and nonhuman primate T stem cell memory cells. Nature Protocols 8(1):33-42
  • Chan LLY, Laverty DJ, Smith T, et al. (2013) Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error. Journal of Immunological Methods 388(1-2):25-32

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