Apotracker™ Green (Apo-15)

Calcium-Independent Apoptosis Probe


Non-antibody probes can be particularly useful in apoptosis detection assays. Annexin V has historically been a gold standard for general apoptosis detection when coupled with another probe for necrosis like impermeant nucleic acid stains (Helix NP™ probes) and esterase substrates (Calcein-AM probes). Annexin V, however, depends on Ca2+ in the staining media. It can also exhibit high background staining and be difficult to incorporate in microscopy applications. Detected in the FITC channel, Apotracker™ Green (Apo-15) is a fluorogenic probe that binds to apoptotic cells in a Ca2+ independent manner, while exhibiting a linear relationship with Annexin V staining, suggesting they are both detecting externalized PS residues. Apotracker™ Green is also useful in microscopy applications on live cells and is retained with paraformaldehyde fixation.

Early Stage-> Mid Stage-> Late Stage Apoptosis

Apotracker™ Green signal is preserved post-fixation

Apotracker™ Green signal is preserved post-fixation

 

Apotracker™ Green (Apo-15) data Day-old murine splenocytes were stained with Apotracker™ Green, washed and then either analyzed unfixed (black) or fixed with FluoroFix™ Buffer (red). Signal could be affected by other harsher fix and permeabilization reagents.

Identify cells in apoptosis by flow cytometry and microscopy

Apotracker™ Green (Apo-15) microscopy image 5-day old HeLa cells were stained with Calcein Red-AM (an indicator of live, healthy cells), Helix NP™ Blue, and Apotracker™ Green (Apo-15).

 

Flow data with Apotracker™ Green (Apo-15) (Left) Day-old splenocytes, gated on Helix NP™ NIR negative cells to exclude necrotic cells. (Right) Helix NP™ NIR-negative cells (red gate, left plot) stained for Apotracker™ Green and BV421™ Annexin V. Staining was performed with Annexin Binding Buffer according to BioLegend protocol. It has not been determined if Annexin V and Apotracker™ Green compete for binding sites.

Apotracker™ Green labels apoptotic, dead cells without the need for specialized buffers

Apotracker™ Green (Apo-15) flow data image Unstimulated (left) and CD95-stimulated (right) Jurkat cells were stained with Apotracker™ Green (Apo-15) and Helix NP™ NIR. Cells were stained for 10-15 min in FACS buffer followed by two washes prior to analysis.
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