Immunochemicals - PKH Dyes - Cell Census Plus

Pre-labeling with PKH26 Dye and Cell Culture.

Wash cells in serum and protein-free medium. Resuspend in Diluent C and add to diluted PKH26 dye for 3 minutes. Adding serum stops the labeling process. After washing in serum containing medium, the cells are ready for culture.

Saturation staining is neither necessary nor desirable in Cell Census Plus. Incorporate only enough dye to generate a brightly fluorescent peak with low CV at time zero. Over-labeling of the cells can generate a loss of membrane integrity, compromise cell recovery and biological function.

A titration optimization for each cell type to be labeled should be performed. For example, a final dye concentration of 2 尋 for 5 x 106 cells in 2 ml is sufficient for human peripheral blood leukocytes and most mouse cell lines.

Immunophenotyping and Flow Cytometry Assay for Proliferation.

Harvest cultured cells and tag with selected fluorochrome conjugated antibody reagents by performing standard immunophenotyping protocols. Using flow cytometry, the cells of interest are analyzed by gating for the phenotype defined by the antibody. Any overlap in emission spectra between PKH26 and FITC or Quantum Red^TM fluorophores can be satisfactorily eliminated or compensated (Figures 5a, 5b, 5c).

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.

Figure 5a, 5b, 5c.
Correlated fluorescence histograms of a three color sample for simultaneous measurement of cell proliferation and subset analysis. See Figure 1 for experimental details. The same list mode data file for Figure 1b is displayed as: 5a - FL-2 (PKH26 fluorescence) versus FL-1 (FITC-CD4)5b - FL-2 versus FL-3 (Quantum Red-CD45RO) 5c - FL-3 versus FL-1

Analysis Using the Cell Proliferation Model.

PKH26 labeled cells have a characteristic fluorescent intensity at time zero. Cell Census Plus assumes fluorescent intensity will reduce by half at each cell division.

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.
Reduced chi-square as a measure of the goodness of fit.

The Proliferation Index is a measurement of proliferative activity. The total number of cells is determined by the summation of the Gaussian areas in each generation (Ak). The original number of cells is calculated by the summation of the Gaussian areas divided by 2k, where k equals the generation number.

Loss of PKH26 dye through the membrane and transfer of dye to unlabeled cells in culture is minimal.

The Simple.mod version of the model assigns the same standard deviation value to all generations.

The Complex.mod version of the model estimates for asymmetrical cell divisions, resulting in widening CV in successive generations.

The standard deviation of initial distribution depends on the uniformity of cell size and the uptake of dye.

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