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

我想用膜联蛋白V标记贴壁细胞，结果发现所有细胞都被标记了，该如何解决这一问题？

Accepted Answer

用胰蛋白酶或其它试剂处理细胞使贴壁细胞分离，会造成膜损伤，从而使这类细胞被膜联蛋白Ⅴ标记。避免出现这一问题的最好方法是将您的细胞放回培养箱中30-45分钟。每隔几分钟涡旋管/板/瓶以防止细胞再次附着。在此恢复期之后，就可以用膜联蛋白V标记您的细胞并进行流式细胞术分析。

Question 2

流式细胞术的优势有哪些？

Accepted Answer

•可测定来自单个细胞的数据。
•可从大量细胞中获得数据，产生细胞群的丰富统计学分析结果。
•由于可测定单个细胞，能够揭示种群的异质性。
•支持多重检测，可鉴定小型亚群。
•可以快速分析数以千计的细胞。
•非常适合血液样本和其他悬浮细胞。
•数据获取后，可以多次重复分析。
•流式细胞仪文件（FCS）可以归档。

Question 3

我能在流式细胞仪上进行哪些应用？

Accepted Answer

可进行多种应用，包括免疫分型、细胞周期分析、凋亡检测（如膜联蛋白V染色检测实验）、CellEvent Caspase-3/7检测、TUNEL检测、细胞活性检测、增殖检测（如CellTrace 检测和Click-iT EdU检测）、MitoProbe检测法测定线粒体电势、利用计数微球进行细胞计数。

Question 4

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 5

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 6

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 7

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, Alexa Fluor™ 680 conjugate, 500 μL - FAQs