Cellometer Vision Application:

Automated Counting and Sizing Freshly Isolated Adipocytes with Minimal Sample Preparation

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Introduction

Brightfield Image of adipocyte sample.
Lipid droplets are indistinguishable
from intact adipocytes.

Fluorescent image showing AO
stained adipocytes. Lipid droplets
are not visible.

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 counting complex samples with minimal sample preparation, such as counting and sizing adipocytes.

The size and number of adipocytes are directly associated with adipose tissue metabolism. However, currently available automatic cell counting methods often require complex sample preparation and use of hazardous reagents. With a traditional manual hemacytometer method, there is a technical challenge in distinguishing intact adipocytes from lipid droplets generated from ruptured adipocytes in the collagenase digestion process. Cellometer Vision incorporates both total cell counting and fluorescence detection in one easy-to-use instrument. By instantly staining enzymatic treated fat tissue samples with acridine orange (AO), a DNA fluorescence dye, Cellometer Vision can detect and analyze fluorescent positive adipocytes. In less than a minute, the system can generate accurate cell counting results and also report cell sizes. Various primary adipocytes, such as human, mice, rat, etc. with diameters of 25um to 250um can be easily loaded into a disposable counting chamber designed especially for adipocytes to eliminate liquid handling reliability issues.

Using proprietary algorithms, Cellometer Vision's robust operating software accurately analyzes cell images, and generates counting data in less than 60 seconds. 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 adipocyte cell count, concentration
and size data.

Method

Direct AO labeling of adipocytes:

Pipette 90µl of adipocyte cell sample into a microtube.
Apply 10µl of 20 µg/ml acridine orange solution to adipocyte cell sample.
Mix cell sample with acridine orange solution gently.
Load 55µl of labeled sample into the PD300 deepwell disposable counting chamber.

Running Assay:

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

Results

Both adipocytes and lipid droplets are visible in the brightfield mode (Figure 1) while AO stained adipocytes are clearly visible in the acquired fluorescence image (Figure 2), distinguishing them from lipid droplets. The software indicates counted cells with green circles (enlarged to show detail). Results display (Figure 3) indicates counted cells, as well as mean size, and automatically calculates concentration.

Figure 1. Brightfield image showing
adipoctyes and lipid droplets.

Figure 2. Fluorescent image showing
counted AO stained adipoctyes.
Lipid droplets are not counted
and indicated by red circle.

Cell size data (Figure 4) and distribution histograms (Figure 5) can be easily generated. All experimental data can be instantly transferred to an Excel spreadsheet or saved in a data table.

Figure 3. Total count (Brightfield) and adipocyte only count
(Fluorescence) as well as adipocyte mean size are indicated
onscreen immediately after image analysis.

Figure 4. Cell diameter data can be easily displayed or exported to
Excel for further analysis.

Figure 5. Adipocyte size distribution histogram displayed on-screen.

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CELLOMETER Vision Trio SPECIFICATIONS:

Imaging Modes: Brightfield & 2 Fluorescence Channels
Filter Set 101: Excitation/Emission Peak: 475nm/535nm
Flter Set 202: Excitation/Emission Peak: 525nm /595nm
Dimensions: 6"x 8.5" x 14" (15cm x 22cm x 36cm)
Weight: 25lbs (11kg)
PC Specs: WinXP/1.8+GHz/1GB RAM/laptop included

Other Cellometer Vision Applications:

Quantifying GFP Transfection
Counting WBCs in Whole Blood without Lysing
Annexin-V for Detection of Apoptosis
Determination of Cell Viability with Propidium Iodide
Counting and Sizing Adipocytes
Counting & Calculating Viability of Hepatocytes
Determining Yeast Viability
Counting and Determining Viability of Stem cells

Join our free webinar. "Improving Cell Counting Data Quality & Throughput" on Friday, March 19, 2010 @ 10:00am EDT. Register now