The ALDEFLUOR™ reagent system is a non-immunological method to identify human stem/progenitor cells on the basis of their aldehyde dehydrogenase (ALDH) activity. It was originally developed for the detection of hematopoietic stem and progenitor cells in human cord blood, bone marrow and mobilized peripheral blood. For detection of ALDH activity in other cell types, different assay conditions may be required to achieve optimal results.

There are different approaches to optimization that we can suggest when utilizing cells that are not of hematopoietic origin. The following suggestions may help to increase the fluorescence intensity of your ALDHbr cells and better discern the ALDHbr population from the negative control:

• Increasing the DEAB concentration – The efficacy of DEAB as an inhibitor differs between cell sources and species. Start by doubling the amount of the inhibitor per reaction. It may be necessary to increase the volume significantly (up to 10 times).
• Optimizing the ALDEFLUOR™ reagent concentration - Test a range of concentrations from 5-fold less to 10-fold more.
• Addition of the DEAB inhibitor prior to the addition of the ALDEFLUOR™ reagent – Prepare 2 tubes (1 control and 1 test) each containing 0.5 mL of cells at the recommended concentration. Add 5 µL of DEAB to the control tube and mix. Then add 2.5 µL of the activated ALDEFLUOR™ reagent to each of the tubes and mix immediately. Certain cell lines have very high ALDH activity which is difficult to inhibit, even in the short amount of time it takes to add the ALDEFLUOR™-reacted cells to the inhibitor. Adding the inhibitor prior to the reaction can help to discern the ALDHbr population in high-expressing cells.
• Optimizing the incubation time - Allow the enzymatic reaction to proceed for less time. We suggest testing 15, 30, 45 and 60 minute incubation times. For example, the SKRB3 mammary cell line has an optimal incubation time between 30 – 45 minutes, incubation for a full hour results in lower fluorescence intensity.
• Optimizing the cell concentration – The reaction is based on the molar concentration of reagents, so hematopoietic cells should be kept at a concentration of 1 x10e6 cells per mL. However, different sample types may produce different results. Decreasing the cell concentration may increase assay signal and increase discrimination of the ALDHbr population. For example, with SKRB3 cells the ALDEFLUOR™ assay results in a much better signal if a cell concentration of 1 – 2 x10e5 cells/mL is used. Because ALDH kinetics across species is not the same, we recommended testing a range of cell concentrations, higher and lower than the concentration recommended for hematopoietic cells.
• Supplementation with additional ABC transporter blockers to inhibit efflux – The ABC transporter blocker already present in the ALDEFLUOR™ buffer may not be optimal for non-human species. Additional ABC transporter blockers to test include verapamil (50 - 100 µM) which may improve blocking in primate species, probenicid (2.5 mM) which may be useful for murine samples and/or 2-deoxy-D-glucose (10 - 100 mM).
• Enrichment of the progenitor population by lineage depletion – In some species and tissues, non-progenitor cells may express high ALDH activity, which masks the ALDH activity of the progenitor population. Removal of lineage-committed cell types (lineage depletion) before assaying with ALDEFLUOR™ will enrich for the progenitor population and enhance the assay read-out.

The specific optimizations required will vary depending on the cell type. Sometimes simply adjusting the cell concentration is sufficient, and other times more than one of these parameters needs to be changed (i.e. decreasing the ALDEFLUOR™ reagent concentration in conjunction with adding the DEAB first).

Please also view our video, Optimization of ALDEFLUOR™ Protocol, for tips on tailoring the ALDEFLUOR™ assay staining procedure to your cell type of interest. For further information on any of the above recommendations please contact Technical Support (techsupport@stemcell.com).