Detection of alkaline phosphatase activity in human immune cells, tumor cells and chick chondrocytes by flow cytometry.

A collaboration with Drs. Steven Doty, Bryan Nestor and Diptendu Rakshit, Hospital for Special Surgery.

Cellular enzymatic activites (such as acid and alkaline phosphatase activity) constitute important metabolic activities in cells and often serve as functional markers for cellular differentiation and tumor status. Enzyme substrates that become fluorescent or modify their fluorescent properties following modification (or non-fluorescent substrates that can modify a fluorescent indicator dye) can be used to detect these enzymatic activities by flow cytometry. We are screening both direct assays (using fluorescent substrates such as 4-methylumbelliferyl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate and fluorescein diphosphate) and indirect assays (using naphthol phosphate substrates and indicator dyes such as Fast Red Violet LB and Fast Red TR) to identify useful assays for detecting alkaline phosphatase activity by flow. These assays can also be combined with other fluorescent parameters (such as fluorescent immunophenotyping or DNA content) to provide even more information. Fluorescent assays for acid phosphatase, NADPH dehydrogenases and other enzymatic processes are also in the works.

(Below.) Alkaline phosphatase activity in HL-60 cells using Fast Red Violet LB (FRV) as an indicator and naphthol bis-phosphate as a phosphatase substrate. This assay is an indirect substrate-indicator system, since dephosphorylation of a non-fluorescent substrate is used to modify a fluorescent indicator dye. Filled and unfilled peaks indicate the presence of FRV with and without substrate respectively, and the inset number is the MFI ratio between peaks. Cells were excited with a 488 nm argon laser and Fast Red Violet fluorescence detected using a 660/20 nm narrow bandpass filter. This assay can be combined with DAPI staining to allow simultaneous detection of DNA content using the dual 488/350 nm argon laser on the Vantage cell sorter

(Below). Alkaline phosphatase activity in HL-60 cells using Fas Red TR as an indicator and naphthol bis-phosphate as a phosphatase substrate, another indirect phosphatase assay. Filled and unfilled peaks indicate the presence of Fast Red with and without substrate respectively. Cells were excited with a 350 nm (ultraviolet) argon laser and Fast Red fluorescence detected using a 424/44 nm narrow bandpass filter.

This assay will be used to assess alkaline phosphatase activity in human monocytes (Dr. Nestor). It will also be used in the microgravity chick chondrocyte project led by Dr. Steve Doty at HSS to assess launch and microgravity effects on chondrocyte development.

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