Kits for Cell Proliferation Analysis

Stock Number Description:

CCPS-1 The Cell Census Plus^TM System

THE CELL CENSUS PLUS^TM SYSTEM, CCPS-1
The Cell Census Plus System is a cell proliferation assay for flow cytometry. Using flow cytometry, Cell Census Plus simultaneously measures cell proliferation of phenotypically distinct subsets of cells within a heterogeneous population.

CELL CENSUS PLUS^TM:
* Provides the number of cell divisions (up to ten generations), relative abundance of each daughter generation, the fraction of non-proliferating cells and the proliferation index.
PLUS
Study distinct cell subsets within a total cell population via immunophenotyping (i.e., antibodies to cell surface markers).
* Measures the endpoint of multistep activation, instead of discrete, early events, such as up regulation of CD69.
* Provides for standardized cytometer setup and normalized cell counts.
* Works with ModFit (sold separately), a computer program, to deconvolute histograms.
* Labels all cell types with a universal fluorescent membrane dye. Membrane labeling is fast, and sufficient for kinetic studies.
* Does not interfere with biological and proliferative activities of cells.
* Uses nonradioactive reagents.

Cell Census Plus contains a red fluorescent dye to label the cell's membrane, microbeads to standardize instrument setup and normalize cell counts and software, the cell proliferation model, to interpret the histogram data.
The Cell Census Plus System takes advantage of a patented fluorescent aliphatic reporter molecule that acts as a membrane dye.(1) Its rapid uptake and stable retention in the plasma membrane and equal distribution into the membranes of the daughter cells make this dye ideal for cell tracking(2-5) and measuring proliferation. Cell labeling does not interfere with cell proliferation. Labeled cells will have a characteristic fluorescence intensity at time zero. Cell Census Plus assumes fluorescent intensity will reduce by one half at each cell division and each daughter cell will retain half the dye present in the mother cell. Therefore, the intensity will be 1/2 in the first daughter generation, 1/4 in the second daughter generation, 1/8 in the third daughter generation, etc.
Cell Census Plus can utilize immuno-gating techniques to measure proliferation of a select subset of cells within the proliferating cell culture.

The Cell Census Plus^TM System
1. Prelabeling of cells with PKH26 dye
2. Cell proliferation in culture
3. Label cells with antibody (Immunophenotyping)
4. Data acquisition on flow cytometer
5. Data analysis using software

The Cell Census Plus process is simple. First, label the entire cell population before exposing it to the proliferation stimulus. The cells are now ready for culture. After proliferation, the cells may be labeled with specific antibodies so that the proliferating activities of specific subpopulations may be studied. Next, ModFit (Verity Software) is used to analyze the data. ModFit is a computer program that deconvolutes raw histogram data into peaks. Each peak represents a generation. The Cell Proliferation Model software provides the parameters for ModFit to perform the histogram deconvolution. It then calculates the proliferation index, a ratio of the total number of cells analyzed versus the calculated number of parental cells required to generate the observed cell number. ModFit and The Cell Proliferation Model program can deconvolute histograms for up to ten generations and give the following numerical values: Number of cell divisions, Percent of cells in each generation for up to 10 generations, Proliferation index based on these percentages, Non-proliferating fraction, and Reduced chi-square as a measure of the goodness of fit.
To add quantitative strength to the results, use the PKH26 reference microbeads to standardize setup of the flow cytometer. Use of these microbeads ensures identical parameters for all assays. PKH26 reference beads are also used to normalize cell counts.

Each Kit Contains:
* 1 vial of PKH26 Dye, a red fluorescent membrane dye excited by the 488 nm argon ion laser found in most flow cytometers. The emission can be detected by using the standard filter setup for phycoerythrin (PE). Each vial of PKH26 dye contains 0.5 ml at 1 x 10-3 M.
* 6 x 10 ml Diluent C, a patented dilution buffer. Cells are resuspended in Diluent C and PKH26 dye is diluted in Diluent C just prior to general surface membrane labeling. Diluent C is supplied sterile.
* 100 ml PKH26 Reference Microbeads, polymer microbeads having fluorescence intensity and light scatter characteristics similar to PKH26-labeled human leukocytes. The reference microbeads are impregnated with PKH26 dye, then carefully formulated as a suspension to serve as a fluorescence intensity and count standard.
* 1 each of The Cell Proliferation Model Software, a computer program for interpreting raw histogram data. The Cell Proliferation Model program runs with ModFit, (Product No. Z35732-4) a DOS program that operates in Windows, and can analyze list mode files from a variety of flow cytometers. ModFit is available from Sigma and Verity Software House, Inc.

References:
1.Horan, P., and Slezak, S., Nature, 340, 167 (1989).
2.Slezak, S., and Horan, P., Blood, 74, 2171 (1990).
3.Samlowski, W., et al., J. Immunol. Meth., 144, 101 (1991).
4.Hugo, P., et al., Nature, 360, 679 (1992).
5.Messina, L., et al., Proc. Nat. Acad. Sci. USA, 89, 12018 (1992).

PKH26 Red Fluorescent Cell Linker technology is covered under U.S. Patent Numbers 4,783,401; 4,762,701; and 4,859,584.

Store at 2-8°C. Shipping Conditions: On wet ice. Please refer to ordering information for details.

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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone:(765) 494-0757; FAX (765) 494-0517; Web http://www.cyto.purdue.edu, EMAIL cdrom3@flowcyt.cyto.purdue.edu

Stock Number	Description:
CCPS-1	The Cell Census Plus^TM System
THE CELL CENSUS PLUS^TM SYSTEM, CCPS-1 The Cell Census Plus System is a cell proliferation assay for flow cytometry. Using flow cytometry, Cell Census Plus simultaneously measures cell proliferation of phenotypically distinct subsets of cells within a heterogeneous population.
CELL CENSUS PLUS^TM: * Provides the number of cell divisions (up to ten generations), relative abundance of each daughter generation, the fraction of non-proliferating cells and the proliferation index. PLUS Study distinct cell subsets within a total cell population via immunophenotyping (i.e., antibodies to cell surface markers). * Measures the endpoint of multistep activation, instead of discrete, early events, such as up regulation of CD69. * Provides for standardized cytometer setup and normalized cell counts. * Works with ModFit (sold separately), a computer program, to deconvolute histograms. * Labels all cell types with a universal fluorescent membrane dye. Membrane labeling is fast, and sufficient for kinetic studies. * Does not interfere with biological and proliferative activities of cells. * Uses nonradioactive reagents.
Cell Census Plus contains a red fluorescent dye to label the cell's membrane, microbeads to standardize instrument setup and normalize cell counts and software, the cell proliferation model, to interpret the histogram data. The Cell Census Plus System takes advantage of a patented fluorescent aliphatic reporter molecule that acts as a membrane dye.(1) Its rapid uptake and stable retention in the plasma membrane and equal distribution into the membranes of the daughter cells make this dye ideal for cell tracking(2-5) and measuring proliferation. Cell labeling does not interfere with cell proliferation. Labeled cells will have a characteristic fluorescence intensity at time zero. Cell Census Plus assumes fluorescent intensity will reduce by one half at each cell division and each daughter cell will retain half the dye present in the mother cell. Therefore, the intensity will be 1/2 in the first daughter generation, 1/4 in the second daughter generation, 1/8 in the third daughter generation, etc. Cell Census Plus can utilize immuno-gating techniques to measure proliferation of a select subset of cells within the proliferating cell culture.
The Cell Census Plus^TM System 1. Prelabeling of cells with PKH26 dye 2. Cell proliferation in culture 3. Label cells with antibody (Immunophenotyping) 4. Data acquisition on flow cytometer 5. Data analysis using software
The Cell Census Plus process is simple. First, label the entire cell population before exposing it to the proliferation stimulus. The cells are now ready for culture. After proliferation, the cells may be labeled with specific antibodies so that the proliferating activities of specific subpopulations may be studied. Next, ModFit (Verity Software) is used to analyze the data. ModFit is a computer program that deconvolutes raw histogram data into peaks. Each peak represents a generation. The Cell Proliferation Model software provides the parameters for ModFit to perform the histogram deconvolution. It then calculates the proliferation index, a ratio of the total number of cells analyzed versus the calculated number of parental cells required to generate the observed cell number. ModFit and The Cell Proliferation Model program can deconvolute histograms for up to ten generations and give the following numerical values: Number of cell divisions, Percent of cells in each generation for up to 10 generations, Proliferation index based on these percentages, Non-proliferating fraction, and Reduced chi-square as a measure of the goodness of fit. To add quantitative strength to the results, use the PKH26 reference microbeads to standardize setup of the flow cytometer. Use of these microbeads ensures identical parameters for all assays. PKH26 reference beads are also used to normalize cell counts.
Each Kit Contains: * 1 vial of PKH26 Dye, a red fluorescent membrane dye excited by the 488 nm argon ion laser found in most flow cytometers. The emission can be detected by using the standard filter setup for phycoerythrin (PE). Each vial of PKH26 dye contains 0.5 ml at 1 x 10-3 M. * 6 x 10 ml Diluent C, a patented dilution buffer. Cells are resuspended in Diluent C and PKH26 dye is diluted in Diluent C just prior to general surface membrane labeling. Diluent C is supplied sterile. * 100 ml PKH26 Reference Microbeads, polymer microbeads having fluorescence intensity and light scatter characteristics similar to PKH26-labeled human leukocytes. The reference microbeads are impregnated with PKH26 dye, then carefully formulated as a suspension to serve as a fluorescence intensity and count standard. * 1 each of The Cell Proliferation Model Software, a computer program for interpreting raw histogram data. The Cell Proliferation Model program runs with ModFit, (Product No. Z35732-4) a DOS program that operates in Windows, and can analyze list mode files from a variety of flow cytometers. ModFit is available from Sigma and Verity Software House, Inc.
References: 1.Horan, P., and Slezak, S., Nature, 340, 167 (1989). 2.Slezak, S., and Horan, P., Blood, 74, 2171 (1990). 3.Samlowski, W., et al., J. Immunol. Meth., 144, 101 (1991). 4.Hugo, P., et al., Nature, 360, 679 (1992). 5.Messina, L., et al., Proc. Nat. Acad. Sci. USA, 89, 12018 (1992).
PKH26 Red Fluorescent Cell Linker technology is covered under U.S. Patent Numbers 4,783,401; 4,762,701; and 4,859,584.
Store at 2-8°C. Shipping Conditions: On wet ice. Please refer to ordering information for details.