Q: I want to study apoptosis using an Annexin V conjugate, but with adherent cells via microscopy instead of flow cytometry. Can this be done?

It has been done, but we dont recommend it. Both healthy cells and apoptotic cells possess phosphatidylserine on the cell surface, which can be detected with Annexin V, but apoptotic cells have significantly more of it. You can easily tell the difference between these two populations with flow cytometry, because flow cytometers are more sensitive and have a higher throughput. But with a microscope, you cannot always tell the difference, especially for adherent cells. Instead, for microscopy, we recommend a different technique, such as detecting caspases with CellEvent Caspase Detection Reagents. Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center .

Question 1

I want to study apoptosis using an Annexin V conjugate, but with adherent cells via microscopy instead of flow cytometry.  Can this be done?

Accepted Answer

It has been done, but we don‘t recommend it. Both healthy cells and apoptotic cells possess phosphatidylserine on the cell surface, which can be detected with Annexin V, but apoptotic cells have significantly more of it. You can easily tell the difference between these two populations with flow cytometry, because flow cytometers are more sensitive and have a higher throughput. But with a microscope, you cannot always tell the difference, especially for adherent cells. Instead, for microscopy, we recommend a different technique, such as detecting caspases with CellEvent Caspase Detection Reagents.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Question 2

I am trying to label adherent cells with annexin V and am finding that everything is getting labeled. How can I fix this?

Accepted Answer

Treating cells with trypsin or other reagents to detach adherent cells causes damage to the membrane, such that cells will be labeled with annexin V. The best way to avoid this problem is to allow your cells to recover for 30-45 min in the incubator. Swirl the tube/plate/flask every few minutes to prevent re-attachment. After this recovery period, you can label your cells with annexin V and analyze by flow cytometry.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Question 3

What are the advantages of flow cytometry?

Accepted Answer

-Measures data from single cells.
-Data are obtained for a large number of cells, generating a rich statistical analysis of cell populations.
-Because single cells are measured, it will reveal heterogeneity within a population.
-With the ability to multiplex, small sub-populations can be identified.
-Thousands of cells can be analyzed rapidly.
-It is ideally suited for blood samples and other cells in suspension.
-Data can be re-analyzed multiple times after acquisition.
-Flow cytometry files (FCS) can be archived.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Question 4

What kinds of applications can I run on a flow cytometer?

Accepted Answer

There are several applications, some of which include immunophenotyping, cell cycle analysis, apoptosis assays such as annexin V staining, CellEvent Caspase-3/7 assay, and TUNEL assay, cell viability, proliferation assays such as CellTrace assay and Click-iT EdU assay, measurements of mitochondrial potential with MitoProbe assays, and cell counting using counting beads.

Find additional tips, troubleshooting help, and resources within our Flow Cytometry Support Center.

Annexin V, biotin-X conjugate, 500 μL - FAQs

I want to study apoptosis using an Annexin V conjugate, but with adherent cells via microscopy instead of flow cytometry. Can this be done?

I am trying to label adherent cells with annexin V and am finding that everything is getting labeled. How can I fix this?

What are the advantages of flow cytometry?

What kinds of applications can I run on a flow cytometer?