Cell Function
Annexin V Experimental Control Groups Setting
Source: Elabscience®Published: Jan 16,2024
Annexin V is a commonly used method to detect apoptosis and usually detected by Flow Cytometry. In order to ensure the accuracy of experimental results and facilitate subsequent data analysis, it is necessary to set up different control groups.
①Sample without autofluorescence:
|
Groups |
Annexin V |
Nuclear Dye |
Sample Type |
|
Blank control |
- |
- |
Cells without any treatment |
|
Biological control |
+ |
+ |
Cells without any treatment |
|
Single Positive control 1 |
+ |
- |
Cells with obvious apoptosis |
|
Single Positive control 2 |
- |
+ |
Cells with obvious apoptosis |
|
Experimental group |
+ |
+ |
Experimental cells |
②Sample with autofluorescence:
|
Groups |
Annexin V |
Nuclear Dye |
Sample Type |
|
Blank Control |
- |
- |
Cells without any treatment and autofluorescence |
|
Biological Control |
+ |
+ |
Cells without any treatment |
|
Single Positive Control 1 |
+ |
- |
Apoptotic cells without autofluorescence |
|
Single Positive control 2 |
- |
+ |
Apoptotic cells without autofluorescence |
|
Single Positive control 3 |
- |
- |
Cells with autofluorescence |
|
Experimental Group |
+ |
+ |
Experimental cells |
Blank Control: Adjust threshold and voltage
Biological Control: Exclude the influence of experimental operation on experimental results and deduct fluorochrome background. This group can also be used as the basis for the experimental group.
Single Positive Control: Adjusting compensation of fluorochrome can also assist in adjusting the voltage of the channel to prevent the signal from exceeding the acceptance range of Flow Cytometry.
Attention:
* Single positive control cells must be used with cells with obvious apoptosis, because the regulatory compensation needs to have enough positive signals.
* The sample with autofluorescence should be set up with three single positive controls to ensure that each tube has only one fluorochrome.
Experimental Group: Detect experimental results
Tips:
The samples are adherent cells:
* The floating cells in the cell culture medium need to be collected and detected together with the adherent cells subsequently collected.
* Avoid cell damage caused by experimental manipulation. The digestion time of pancreatic enzymes should be appropriate, and the operation should be gentle when digesting the adherent cells.
* Do not use pancreatic enzymes containing EDTA, as EDTA affects the binding of Annexin V to PS. If EDTA-containing pancreatic enzymes must be used, please wash the cells after termination of digestion to remove EDTA as much as possible.
* When the cells are difficult to digest, they can be digested in batches to avoid overdigestion and leading to false positive results.
