Jurkat cells were used to analyze cell cycle kinetics following treatment with the cell-cycle-arresting drug etoposide. Etoposide is designed to arrest the cells at the G2 phase of the cell cycle. Jurkat cells were incubated with media only (control) or etoposide (0.06 µM, 0.12 µM) for 24 hours. Control and drug-treated cells were ethanol fixed and stained with cell cycle propidium iodide reagent. For each sample, 20 µl of cell sample (at ~4 x 106 cells / mL) was loaded into a Cellometer imaging chamber, inserted into the Vision CBA Analysis System, and imaged in both bright field and fluorescence. The fluorescence intensity for each cell was measured and a cell cycle histogram is automatically generated for each sample using the optimized Nexcelom cell cycle data layout in FCS Express 4 Flow Software. Gating can be manually optimized directly on the histogram with automatic update to the associated data table.
The Cellometer fluorescence-based cell cycle analysis can be effectively implemented in studies examining the efficacy of cell cycle arresting drugs.
One of the most common and popular methods for cell cycle detection is the use of fluorescence-based dyes. There are a number of fluorescent-based dyes that are capable of binding to double stranded DNA upon cell fixation. Propidium iodide (PI) and DAPI are two such dyes. Since the amount of bound fluorescent dye is directly proportional to the amount of DNA present within a cell, these dyes can be used to detect the cell cycle within a population of cells.
How it works:
Since the amount of DNA doubles from 2n to 4n between G1 and G2/M phases, and the amount of PI incorporated is correlated to the amount of DNA within each cell, we can generate a histogram based on PI fluorescence intensity.
The Cellometer instrument acquires a bright-field and a fluorescent image for each sample tested. The bright-field image allows researchers to verify cell morphology, evaluate the degree of homogeneity of the sample, and identify the presence of cellular debris
The fluorescent counted image can be used to confirm that cells are counted correctly. Individual counted cells are outlined in green. Uncounted cells are outlined in yellow. Cellometer software uses proprietary algorithms to accurately count individual cells within clumps.
Once counting is complete, a cell cycle histogram is automatically generated for each sample using the optimized Nexcelom cell cycle data layout in FCS Express 4 Flow Software. Gating can be manually optimized directly on the histogram with automatic update to the associated data table.
Representative data set:
In this example, untreated Jurkat cells were used to analyze cell cycle kinetics. The cells were ethanol fixed, stained with propidium iodide cell cycle reagent, and 20 µl of cell sample was loaded into a Cellometer counting chamber. The counting chamber was inserted into the Vision CBA Analysis System, and imaged in both bright field and fluorescence. The fluorescence intensity histogram and associated data table was generated in FCS Express 4 software.
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 counting white-blood-cells in whole blood.
Acridine orange (AO) is a fluorescent nucleic acid stain that has been used to measure nucleated cell concentration in various cell types, including mammalian cell lines, mammalian primary cells, and yeasts. AO is a membrane-permeable dye that stains nucleated cells green.
The following article describes a simple and specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns.
Author institutions: Translational Immunology Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, and Translational Autoinflammatory Disease Section, Office of the Clinical Director, National Institute of Arthritis and Musculoskeletal and Skin Diseases
Authors: Barbara Yang, Tuyet-Hang Pham, Raphaela Goldbach-Mansky, Massimo Gadina
Journal: J Vis Exp. 2011; (49): 2662.
A key component of this assay is to standardize the dose response to the number of cells stimulated. They have found 2×106 cells /mL is the sufficient concentration for optimal and measureable concentration of IL-1beta and other cytokines.
“Using Cellometer Vision, diluted blood can be counted without lysing RBC y choosing the whole blood count option using acridine orange (AO). Add one part diluted blood with one part AO and load 20 ml of the 1:1 mixture into a disposable counting chamber. The final AO concentration after dilution should be 1mg/mL. Adjust accordingly with incomplete RPMI 1640 media to final cell centration of 2×106 cells /mL.”
Cellometer Vision has a convenient cell concentration adjustment calculator. Input the original concentration of 20mL, based on the measured cell concentration for each sample, the software calculates the adjustment required to obtain 2×106 cells /mL.
Accurate Measurement of Peripheral Blood Mononuclear Cell Concentration using Image Cytometry to Eliminate RBC-Induced Counting Errors
Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research.
Ten Questions to Ask Before Purchasing an Automated Cell Counter
Believe it or not, all cell counters are not created equally. Ask a colleague with a handheld counter that sits in a drawer or a bench-top cell counter that acts as a paperweight and they will tell you that some cell counters don’t work as expected. Before purchasing an automated cell counter, ask the representative the following questions. Knowing these answers will help ensure that you purchase the right cell counter for your applications. (more…)
Rapid Quantification of Pathogenic Fungi by Cellometer Image-Based Cytometry
Joint collaboration between Nexcelom Bioscience and Merrimack College
Objective: To demonstrate a simple image cytometry method for the quantification of viable pathogenic fungi, Histoplasma capsulatum. To validate image cytometry as a viable alternative method to CFU counting, a method that is time-consuming (sometimes taking 1-2 weeks for the formation of visible colonies), labor-intensive, and limited in efficiency and sensitivity. To validate image cytometry as a viable alternate to flow cytometry, due to the high cost, complexity, and biosafety containment issues associated with the use of flow cytometry for analysis of pathogenic fungi. (more…)
Calcein AM (Calcein acetoxymethyl ester) is a non-fluorescent compound that passively enters cells. In metabolically active cells, Calcein AM is converted by cytosolic esterases into green fluorescent Calcein. The fluorescent Calcein is retained by live cells with intact membranes. Only cells possessing active cytosolic esterases fluoresce green. This allows for quick and easy detection of metabolically-active (vital) cells in a sample. (more…)
Review: Unlicensed NK Cells Target Neuroblastoma Following Anti-GD2 Antibody Treatment
Author: Tarek, N., et al.
J. Clin. Invest. 2012; 122(9): 3260-3270. doi:10.1172/JCI62749.
Background: Neuroblastoma (NB) is the most common childhood extracranial solid tumor. Nearly two-thirds of diagnosed patients exhibit poor long-term survival despite aggressive treatment approaches. Treatment with monoclonal antibodies against the disialoganglioside surface antigen GD2 has resulted in lower recurrence and improved survival. The anti-GD2 mAb 3F8 utilizes antibody-dependent cell-mediated toxicity (ADCC) via myeloid and natural killer (NK) cells to kill the neuroblastoma. (more…)
Natural Killer (NK) Cells are large granular lymphocytes that belong to the innate immune system and make up approximately 10% of circulating lymphocytes. Unlike T cells, NK cells do not express CD3. NK cells are critical for protection from life-threatening infections and are important mediators of antitumor immunity. Rare reports of complete NK-cell deficiencies in humans have resulted in fatal infection during childhood. Uncontrolled or inappropriate NK cell response can lead to pathological conditions such as allograft rejection, graft vs. host disease, diabetes, aplastic anemia, and various autoimmune and neurological diseases. (more…)
GFP and Determination of Transfection Efficiency
Green Fluorescent Protein (GFP) is a 26.9 kDa protein that fluoresces bright green when exposed to blue or ultraviolet light. GFP was first identified as a protein and extracted from the Aequorea victoria jellyfish in 1962 by Osamu Shimomura, et al. The GFP protein was first cloned in 1992 and it was soon confirmed that GFP protein expressed in other organisms generates fluorescence. An area within a cell or tissue is briefly illuminated, causing the GFP protein to fluoresce, allowing GFP-tagged proteins to be identified. In 2008, Osamu Shimomura, Marty Chalfie, and Roger Tsien received the Chemistry Nobel Prize “for the discovery and development of the green fluorescent protein.” In recent years, various mutations have yielded forms of GFP offering improved brightness, a modified excitation and emission spectra, improved 37°C folding, reduced aggregation at high concentrations, and improved diffusibility inside cells. Today, GFPs are used in an increasing variety of cell-based assays to measure intra-cellular events. GFP genes are co-transfected with genes of interest to measure transfection efficiency, GFP fusion tags are used to identify the localization and fate of normal host proteins, and specifically-engineered GFPs are used as indicators of phosphorylation.
With the Cellometer Vision Image Cytometry Systems, researchers can easily quantify the percentage of GFP-positive cells within a population, allowing for fast, accurate determination of transfection efficiency. Researchers can overlay histograms for control and treated samples for advanced gating of GFP- and GFP+ cell populations.
1Tsien, Roger Y., (1998). The Green Fluorescent Protein, Annu. Rev. Biochem. 67:509-44.
2http://www.conncoll.edu/ccacad/zimmer/GFP-ww/timeline.html,accessed September 25, 2012.