A flow cytometer equipped with a 488 nm laser and a 635 nm laser for excitation and appropriate filters for detecting APC, PE, PE-Cyanine5 and 7-AAD fluorescence is required for use.
• Optimized to detect ALDHbrCD34+ cells in CB
• Enumerates viable, metabolically active, primitive cells in the CD34+ cell population
- 1 x ALDEFLUOR™ Kit (Catalog #01700)
- ALDEFLUOR™ Reagent
- ALDEFLUOR™ DEAB Reagent
- ALDEFLUOR™ Assay Buffer
- 2 N HCl
- 2 x CD34+ Cell Detection Kits
- 7-AAD Viability Dye
- Anti-Human CD45 Antibody, Clone HI30, PE
- Anti-Human CD34 Antibody, Clone 581, APC
- Anti-Human CD235ab (Glycophorin AB) Antibody, Clone HIR2, PE-Cyanine5
This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.
Data and Publications
Figure 1. General ALDHbr Assay Procedure
For a detailed protocol please refer to the ALDHbr Assay Kit Product Information Sheet (PIS)
Figure 2. Gating Strategy to Identify 7-AAD-GlyAB-CD45+ALDHbrCD34+ Cells
CB mononuclear cells (106 cells/mL) were stained with the ALDHbr Assay Kit and analyzed on a flow cytometer equipped with 488 nm and 625 nm lasers for detecting ALDEFLUOR™ (BAA), 7-AAD, APC, PE and PE-Cyanine5 fluorescence. The gating strategy for identifying (A) 7-AAD-GlyAB-CD45+ALDHbrCD34+ cells is shown using a fully stained sample (Sample) as described above. Staining of a (B) DEAB negative-control sample (Negative Control) is used to verify the placement of the ALDHbr gate. Gold lines depict the gates used at each stage to identify the desired cell types. Note: The gating of ALDHbr cells is greatly simplified if red blood cells (RBCs) are first removed from CB samples, prior to staining with ALDEFLUOR™. The ErythroClear™ Red Blood Cell Depletion Kit (Catalog #01739) is designed for the depletion of RBCs from up to 16 small volume CB samples.
Figure 3. The Frequencies of CD34+, ALDHbr and ALDHbrCD34+ Cells in CB Samples are Correlated with Colony Numbers as Measured in CFU Assays
CB mononuclear cells (n=23) were split into two parts. Half of each sample was stained with the ALDHbr Assay Kit and analyzed, as described in Figure 1. The percentages of (A) CD34+, (B) ALDHbr and (C) ALDHbrCD34+ cells in the 7-AAD-GlyAB-CD45+ population in each sample was determined. The other half of each sample was plated in a CFU assay using MethoCult™ Classic H4434 medium (Catalog #04434) and colonies were counted 14 days later. The frequency of CFUs was determined as a percentage of viable total nucleated cells (TNCs) for each sample. The table in (D) shows the number of cells from each subpopulation that contains 1 CFU. The data show that the frequency of ALDHbrCD34+ cells is highly correlated with the frequency of functional progenitor cells (R2: coefficient of determination).
Figure 4. The Percentage of Viable CD45+ Cells that Express CD34+ and/or ALDHbr Vary Among Different CB Samples
CB mononuclear cells (106 cells/mL) were stained and analyzed as described (Figure 1). Shown are the percentages of cells in the 7-AAD-GlyAB-CD45+ gated population that are CD34+, ALDHbr and ALDHbrCD34+. Each column represents the mean ± S.D. of three replicate assays for each CB sample (n=7).
Figure 5. The Analysis of the ALDHbrCD34+ Cell Subset is Consistent Across Different Flow Cytometers
A single CB mononuclear cell sample (106 cells/mL) was split into 3 parts, each of which was separately stained with the ALDHbr Assay Kit (see Figure 1 for protocol) and separately analyzed on three different flow cytometry instruments; a FACScan DXP6, FACSCalibur and Accuri C6. All instruments were equipped with 488 nm and 635 nm lasers for detecting ALDEFLUOR™ (BAA), 7-AAD, APC, PE and PE-Cyanine5 fluorescence. The frequency of ALDHbrCD34+ cells in each sample was determined as a percentage of the 7-AAD-GlyAB-CD45+ cell population measured by each instrument. There was no significant difference in the number of ALDHbrCD34+ cells measured by these flow cytometers (p > 0.05). Each column represents the mean ± S.D. for three replicates of the analyzed CB sample evaluated on each flow cytometer.