TITLE: Isolation and identification of cancer stem cells by ALDH activity assay 
AUTHORS: Kwangok  P Nickel, Austin M. Maas, Andrea Pennati, Randall Kimple

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cancer stem cells (CSCs) are a small subpopulation of tumor cells that are thought to be responsible for recurrence and metastasis of cancer due to their ability for self-renewal and differentiation into multiple cancer cell types. </div><div class = "text-block">Multiple methods have been used to identify CSCs using flow cytometry.  Cell surface markers such as CD44 and CD133 can be used to identify the CSC population in tumor cells. Also, they can be isolated from a heterogeneous population using an assay that tests for the aldehyde dehydrogenase (ALDH) activity within the cell. </div><div class = "text-block">ALDH activity is measured by flow cytometer and expressed by percentage. A higher percentage indicates more CSCs are present in the sample. Aldehyde hydrogenases (ALDHs) are a group of enzymes that catalyze the oxidation of aldehydes to carboxylic acids. They are involved in detoxification of aldehydes produced by metabolic processes or cytotoxic drugs. An increase in ALDH activity may indicate cells are more resistant to chemotherapy. In addition, high ALDH activity has been shown in CSCs of different cancer types and is now considered to be a standard marker of CSCs. The Aldefluor assay has been used to identify and isolate cells with high ALDH activities. This assay is based on the principle that ALDH can convert the ALDH-substrate, Bodipy-aminoacetaldehyde (BAAA) into Bodipy-aminoacetate (BAA) which is retained inside the cells. BAAA is uncharged and can diffuse freely into intact viable cells, however BAA can not cross the membrane due to its net negative charge, which makes remain in the cells and the assay buffer prevents efflux of the BAA from the cells. Therefore, the amount of BAA fluorescence in cells is proportional to ALDH activity and can be measured using a flow cytometer. A specific inhibitor of ALDH, diethylaminobenzaldehyde (DEAB), is used for background fluorescence control. </div></div>

[STEPS]
?. Prepare activated ALDEFLUOR assay reagent, Bodipy-aminoacetaldehyde (BAAA), according to the manufacturer’s directions. Make  aliquots and store them at  .Note:  In this example, a head and neck cancer cell line, UM-SCC47 , is analyzed. The experimental approach can be used to study cells isolated from primary human cancers or with established cancer cell lines.  Also, Attune NxT (Thermo Fisher Scientific) is used as a choice of flow cytometer.
50 µl
-20 °C
?. Trypsinize UM-SCC47 cells and count cells using a hemocytometer or automated cell counter. Cell numbers should be at least 3x 106 to prepare the following  6 tubes.UnstainedPI onlyTest (ALDH)Control (DEAB)Test (ALDH) without PI: FITC onlyControl (DEAB) without PI: FITC only
?. Centrifuge cells at  for  5 min to pellet, remove the supernatant and resuspend in ALDEFLUOR assay buffer to the final concentration of 1 x106 cell/ml in the both “Test” tubes.  Prepare each Unstained and PI tube with 0.5 X 106 cells with 0.5 ml ALDEFLUOR assay buffer.
Centrifuge: 250 34
?. Add  of diethylaminobenzaldehyde (DEAB), reagent provided as part of the ALDEFLUOR assay kit to both “Control” tubes.  Close the tube immediately to prevent the evaporation of the DEAB solution (95% ethanol).
5 µl
?. Add  of activated ALDEFLUOR reagent to both “Test” tubes and mix by gentle vortexing or inversion.
5 µl
?. Immediately transfer  of mixture from each ”Test” tube to each “Control” tube.
0.5 ml
?. Incubate both “Test” and “Control” tubes for 45 min at   in the dark. Do not exceed 60 min (incubation time should be optimized for cell type).
37 °C
?. Following incubation, gently pellet cells by centrifugation at  for 5 min.
Centrifuge: 250 34
?. Remove supernatant and resuspend cells in  of ALDEFLUOR assay buffer. Transfer to 5 ml flow tubes and store the cells on ice or at in the dark.
0.5 ml
4 °C
?. Add  of 1 mg/ml PI to PI, “Test “and “Control” tubes prior to flow analysis. Do not add PI to “Test” and “Control” FITC only tubes.
1 µl
?. Perform flow cytometry.
?. Launch the Attune NxT software.
?. Create an Experiment.On the main menu, click New Experiment.Select Experiment type and enter Experiment name.
?. CompensationCompensation setup is needed due to the spillover of FITC into PI caused by overlapping spectrum of FITC (ALDH positive cells will be detected in this channel) to PI.Click Compensation Setup and select the source of compensation controls (BL1 for FTIC and BL2 for PI), the measurement parameter, background fluorescence mode and compensation parameters.Optimize FSC (Forward Scatter) and SSC (Side Scatter) Voltage and Threshold settings for Unstained sample.  Optimize Fluorescence channel settings for FITC and PI with FITC and PI only samples.     Record compensation.Optimize FSC (Forward Scatter) and SSC (Side Scatter) Voltage and Threshold settings for Unstained sample.Optimize Fluorescence channel settings for FITC and PI with FITC and PI only samples.
?. Run sample.Set up for 5 dot plots.Run “Control (DEAB)” sample to set the gate of the ALDH positive population using the rightmost edge of the stained the “Control” DEAB population. This permits to identify the negative cells with no ALDH activity (ALDH-) and the positive cells with ALDH activity (ALDH+).Place the corresponding ALDH “Test” tube into the flow cytometer.  Collect 100,000 events in live single cells gate.
?. In this figure, A: the Gate is set on the DEAB treated sample in order to include a small number of cells as ALDH positive (4.14%).  B: a representative plot for UM-SCC47 cells is shown and demonstrates 39.09% of the population is ALDH-positive. We typically perform a minimum of 3 biologic replicates (3 independent experiments) and average the present positive cells from the 3 replicates. When this approach is taken, the mean and standard deviation should be reported.