Rapid Detection of Apoptosis in Jurkat Cells with FITC Conjugated Annexin-V

Cellometer Vision

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Introduction

Brightfield image of Jurkat cells.

Fluorescent image of Jurkat cells.
White spots indicate FITC labeled
annexin-V.

Cellometer Vision incorporates image based cell counting and fluorescence detection in a compact and easy-to-use instrument. With fluorescence detection capabilities, Cellometer Vision is an ideal solution for many complex cell population characterization assays such as rapidly detecting apoptotic cells with FITC conjugated annexin-V.

Apoptosis is programmed cell death that plays a critical role in organism homeostasis and tissue development. Abnormal apoptosis processes have been associated with diseases including various tumors. There are a number of in-vitro and in-vivo assays for detecting apoptosis cells. Fluorescein FITC conjugated annexin-V binding assay is one of the simplest methods to detect the early stage of apoptosis. Traditionally, flow cytometry is used to quantitatively analyze apotosis, however, due to complexity, cost and availability limitations, they are often not the best solution for rapid and routine cell characterizations.

Cellometer Vision was developed to count total cells and identify FITC-annexin-V positive apoptotic cells based on the microscopic and fluorescence characteristics of the cell sample. Detection typically takes less than 60 seconds and only requires 20µl of cell sample.

Cell images and all analysis data, including cell size distribution histograms, can be saved for documentation. Data can also be easily exported to Microsoft Excel spreadsheets for further analysis.

Counting results box displays total and fluorescence cell count,
percentage of apoptotic cells, mean size & concentration.

Method

Labeling of cell surface with FITC conjugated annexin-V

Harvest Jurkat cells both induced with anti-CD95L antibody (EOS9, Biolegend, San Diego, CA) and uninduced.
Spin down at 1800 rpm for 5 minutes.
Wash the cells one time with apoptosis wash buffer(AWB).
Discard the supernatant.
Re suspend the cells in AWB buffer at concentration of ~5X10⁶/ml.
Label reaction tubes & add 0, 50, 25, and 12.5µL of FITC conjugate annexin-V (40µg/ml) into each tube and apply 50, 0, 25, and 37.5µl of AWB buffer into each tube accordingly.
Add 100µl of Jurkat cells into each tube and mix well by briefly vortexing.
Incubate the tubes in the dark at room temp for 15 min.
Wash the cells twice with AWB.
Re suspend cells with 0.25ml of AWB and mix gently.

Running Assay:

Load 20µl of labeled sample into the disposable counting chamber.
Insert counting chamber into Cellometer Vision
Select assay from drop-down menu and enter sample ID
Preview cell images and click 'Count' to begin analyzing sample
Review images and counting results.
Save or export images and/or report data.

Results

Total counted Jurkat cells are indicated by green circles in the brightfield image (Figure 1). Apoptotic cells are indicated as fluorescent positive, green circled cells in the fluorescence image (Figure 2). Total and fluorescence positive cells counted, concentration, mean size, and percentage are displayed in the results box (Figure 3).

Figure 1. Total counted cells are indicated
in the brightfield image by green circles

Figure 2. Apoptoitc cells are indicated
by green circles.

Cell & fluorescent size distribution histograms, scatter plots (figure 5) & data files can be instantly generated and exported to an Excel spreadsheet or saved in a data table.

Figure 3. Total Concentration (Brightfield), Apoptotic cell concentration
(Fluorescence) and % positive are displayed immediately after image analysis