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Apoptosis detection kit for Annexin V cell apoptosis
NegotiableUpdate on 05/06
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Overview
The Annexin V-APC Cell Apoptosis Detection Kit is a Ca2+- dependent phospholipid binding protein with a molecular weight of 35-36kD, which can specifically bind to PS that flips out of the membrane during cell apoptosis. PS eversion occurs before cell nucleus rupture, DNA fragmentation, and the appearance of apoptosis related proteins, which makes the binding of Annexin V to PS early in apoptosis .....
Product Details
Annexin V apoptosisIntroduction to Test Kit
Storage temperature:2-8 ℃, avoid light
Annexin V apoptosisTest kitPrecautions
Long term use can be stored at -20 ℃.
2. Avoid repeated freezing and thawing of Annexin V-APC staining solution. Repeated freeze-thaw cycles can lead to failure.
When Annexin V-APC staining solution needs to be stored for a long time, it can be packaged in small quantities according to usage and stored at -20 ℃.
4. Please use up the reagents as soon as possible after unpacking!
2. Avoid repeated freezing and thawing of Annexin V-APC staining solution. Repeated freeze-thaw cycles can lead to failure.
When Annexin V-APC staining solution needs to be stored for a long time, it can be packaged in small quantities according to usage and stored at -20 ℃.
4. Please use up the reagents as soon as possible after unpacking!
Validity period
1 year
Testing method
1. Flow cytometer
2. Laser confocal microscopy
3. Fluorescence microscope
2. Laser confocal microscopy
3. Fluorescence microscope
Applicable samples
1. Suspended cells
2. Adherent cells
2. Adherent cells
Product Features
1. Convenience: Can be detected using flow cytometry or fluorescence microscopy;
2. Quick: The experiment can be completed within 1 hour;
3. Accuracy: Able to accurately distinguish between live cells, apoptotic cells, and necrotic cells, and count the proportion of each group of cells.
2. Quick: The experiment can be completed within 1 hour;
3. Accuracy: Able to accurately distinguish between live cells, apoptotic cells, and necrotic cells, and count the proportion of each group of cells.
Product application
1. Flow cytometer
2. Laser confocal microscopy
3. Fluorescence microscope
2. Laser confocal microscopy
3. Fluorescence microscope
Instrument preparation
1. Flow cytometry, fluorescence microscope, or laser confocal microscopy
2. Centrifuge
3. Pipette
4. Refrigerator
5. Ice box
2. Centrifuge
3. Pipette
4. Refrigerator
5. Ice box
Reagent preparation
1. PBS buffer (pH 7.4, commonly used 10mM phosphate buffered saline solution (1X) in the laboratory)
2. Or HBSS (Hank's Balanced Salt Solution)
2. Or HBSS (Hank's Balanced Salt Solution)
Preparation of consumables
1. Centrifugal tube
2. Suction head
3. Disposable gloves
4. Aluminum foil
2. Suction head
3. Disposable gloves
4. Aluminum foil
Annexin V apoptosisTest kitPrecautions for use
1. Before opening the screw cap micro reagent tube, please briefly centrifuge the reagent to collect the liquid on the inner wall of the cap to the bottom of the tube, in order to avoid insufficient reagent volume due to liquid loss when opening the cap.
2. Cell processing requires careful handling to avoid human damage to cells as much as possible. Centrifugal force should be minimized as much as possible without losing cells, and the action of resuspending cells should be gentle to avoid repeated intense blows. No need for vortex oscillator.
Annexin V-FITC (etc.) and Propidium iodide (etc.) are photosensitive substances. Please avoid light during operation and minimize their exposure time to light. If necessary, use a covered ice box or aluminum foil to avoid light. After labeling, keep the cells in a dark environment. Short exposure to light during the pipetting process (<30 seconds) is acceptable.
4. As apoptosis is a rapid and dynamic process, analysis should be conducted immediately after staining.
2. Cell processing requires careful handling to avoid human damage to cells as much as possible. Centrifugal force should be minimized as much as possible without losing cells, and the action of resuspending cells should be gentle to avoid repeated intense blows. No need for vortex oscillator.
Annexin V-FITC (etc.) and Propidium iodide (etc.) are photosensitive substances. Please avoid light during operation and minimize their exposure time to light. If necessary, use a covered ice box or aluminum foil to avoid light. After labeling, keep the cells in a dark environment. Short exposure to light during the pipetting process (<30 seconds) is acceptable.
4. As apoptosis is a rapid and dynamic process, analysis should be conducted immediately after staining.
5. The use of Annexin V-FITC (etc.) assay kit to detect apoptosis requires targeting live cells. Do not fix cells, as fixing operations can interfere with the results.
Usage method
Sample staining:
1. Collect suspended cells by centrifugation. Centrifuge at 300 × g-500 × g force, 2-8 ° C, centrifuge for 5 minutes, discard the culture medium.
Wash the cells twice with cold PBS. (300 × g-400 × g, 2-8 ° C, centrifuge for 5 minutes to collect cells).
3. Suspend cells in 100 μ l of 1X Annexin V binding solution at a concentration of approximately 1-5 X 106 cells/ml.
4. Add 5 μ l Annexin V-APC staining solution to the cell suspension, gently mix well, and incubate at 2-8 ° C in the dark for 5-15 minutes.
5. After adding PI (or 7-AAD) staining solution, gently mix and incubate at 2-8 ° C in the dark for 1-3 minutes. (This step can be selected, and according to experimental needs, only APC single staining can be performed).
6. Add 400 μ l PBS or 1X Annexin V binding solution and mix gently.
7. Immediately use a flow cytometer for detection.
Flow cytometry analysis:
The processed cells can now be analyzed on a flow cytometer.
Follow the conventional procedure for flow cytometry apoptosis detection.
The fluorescence excitation wavelength of Annexin V-APC is 652 m and the emission wavelength is 670 m; the fluorescence excitation wavelength of PI is 535 m and the emission wavelength is 620 m.
Before conducting on-site testing, it is necessary to prepare a quality control sample using the cells to be tested. The fluorescence compensation of the flow cytometer and the range of the cross gate should be set accordingly
(I) Cells not transfected with GFP or other markers
① Blank tube: negative control group cells, unstained cells. No Annexin V/APC, PI added. Used to regulate voltage.
② Single staining tube: For cells with obvious apoptosis, only Annexin V/APC staining is added. Used for adjusting compensation.
③ Single staining tube: For cells with obvious apoptosis, only PI staining is added. Used for adjusting compensation
④ Detection tube: Add Annexin V/APC and PI to the processed cells to be tested. After adjusting the voltage compensation with a blank tube and a single anode tube, obtain the required flow data.
(II) Transfect GFP cells
① Untransfected blank tubes: Untransfected cells, negative control group cells, unstained cells. No Annexin V/APC, PI added. Used to regulate voltage.
② Transfect GFP blank tube: Control group cells transfected with GFP, unstained cells. No Annexin V/APC, PI added. Used for adjusting compensation
③ Untransfected single staining tube: Untransfected cells with obvious apoptosis were stained only with Annexin V/APC. Used for adjusting compensation.
④ Untransfected single staining tube: For cells that have not been transfected and show obvious apoptosis, only PI staining is added. Used for adjusting compensation
⑤ Detection tube: Add Annexin V/APC and PI to the processed cells to be tested. After adjusting the voltage compensation with a blank tube and a single anode tube, obtain the required flow data.
1. Collect suspended cells by centrifugation. Centrifuge at 300 × g-500 × g force, 2-8 ° C, centrifuge for 5 minutes, discard the culture medium.
Wash the cells twice with cold PBS. (300 × g-400 × g, 2-8 ° C, centrifuge for 5 minutes to collect cells).
3. Suspend cells in 100 μ l of 1X Annexin V binding solution at a concentration of approximately 1-5 X 106 cells/ml.
4. Add 5 μ l Annexin V-APC staining solution to the cell suspension, gently mix well, and incubate at 2-8 ° C in the dark for 5-15 minutes.
5. After adding PI (or 7-AAD) staining solution, gently mix and incubate at 2-8 ° C in the dark for 1-3 minutes. (This step can be selected, and according to experimental needs, only APC single staining can be performed).
6. Add 400 μ l PBS or 1X Annexin V binding solution and mix gently.
7. Immediately use a flow cytometer for detection.
Flow cytometry analysis:
The processed cells can now be analyzed on a flow cytometer.
Follow the conventional procedure for flow cytometry apoptosis detection.
The fluorescence excitation wavelength of Annexin V-APC is 652 m and the emission wavelength is 670 m; the fluorescence excitation wavelength of PI is 535 m and the emission wavelength is 620 m.
Before conducting on-site testing, it is necessary to prepare a quality control sample using the cells to be tested. The fluorescence compensation of the flow cytometer and the range of the cross gate should be set accordingly
(I) Cells not transfected with GFP or other markers
① Blank tube: negative control group cells, unstained cells. No Annexin V/APC, PI added. Used to regulate voltage.
② Single staining tube: For cells with obvious apoptosis, only Annexin V/APC staining is added. Used for adjusting compensation.
③ Single staining tube: For cells with obvious apoptosis, only PI staining is added. Used for adjusting compensation
④ Detection tube: Add Annexin V/APC and PI to the processed cells to be tested. After adjusting the voltage compensation with a blank tube and a single anode tube, obtain the required flow data.
(II) Transfect GFP cells
① Untransfected blank tubes: Untransfected cells, negative control group cells, unstained cells. No Annexin V/APC, PI added. Used to regulate voltage.
② Transfect GFP blank tube: Control group cells transfected with GFP, unstained cells. No Annexin V/APC, PI added. Used for adjusting compensation
③ Untransfected single staining tube: Untransfected cells with obvious apoptosis were stained only with Annexin V/APC. Used for adjusting compensation.
④ Untransfected single staining tube: For cells that have not been transfected and show obvious apoptosis, only PI staining is added. Used for adjusting compensation
⑤ Detection tube: Add Annexin V/APC and PI to the processed cells to be tested. After adjusting the voltage compensation with a blank tube and a single anode tube, obtain the required flow data.
Common problem analysis
During the experiment, Annexin V did not stain or had a low positivity rate. Possible reasons are as follows:
1. Improper setup of flow cytometer/microscope used for detection. Adjust and use the correct instrument settings.
2. Determine whether the inducer in the experiment can induce apoptosis. This situation can be ruled out by setting a positive drug control with precise apoptosis induction effect.
3. The cells have not initiated apoptosis. Redefine the time point for initiating apoptosis after induction (time course experiment).
4. The number of cells is relatively small. Increase the number of cells.
5. Improper digestion of adherent cells. The binding of Annexin V to PS requires Ca2+ions, and the binding solution contains Ca2+ions. The digestion of EDTA can affect staining. It is recommended to use EDTA free or wash with PBS after digestion.
The positive rate was relatively high during the experiment. Possible reasons are as follows:
1. Improper setup of flow cytometer/microscope used for detection. Adjust and use the correct instrument settings.
2. The vitality of the cells themselves is too poor. In the experiment, it was found that the proportion of AnnexinV+/PI+double positive cells in control cells without induced apoptosis after staining was too high. The reason for this result may be due to the poor vitality of the cells themselves. It is recommended to use trypan blue staining to calculate cell viability, and cells that reject trypan blue staining should be greater than 95%. If the vitality is low, it is recommended to revive the cells again. Usually, newly revived cells need to be passaged at least 3 times before experiments can be conducted.
1. Improper setup of flow cytometer/microscope used for detection. Adjust and use the correct instrument settings.
2. Determine whether the inducer in the experiment can induce apoptosis. This situation can be ruled out by setting a positive drug control with precise apoptosis induction effect.
3. The cells have not initiated apoptosis. Redefine the time point for initiating apoptosis after induction (time course experiment).
4. The number of cells is relatively small. Increase the number of cells.
5. Improper digestion of adherent cells. The binding of Annexin V to PS requires Ca2+ions, and the binding solution contains Ca2+ions. The digestion of EDTA can affect staining. It is recommended to use EDTA free or wash with PBS after digestion.
The positive rate was relatively high during the experiment. Possible reasons are as follows:
1. Improper setup of flow cytometer/microscope used for detection. Adjust and use the correct instrument settings.
2. The vitality of the cells themselves is too poor. In the experiment, it was found that the proportion of AnnexinV+/PI+double positive cells in control cells without induced apoptosis after staining was too high. The reason for this result may be due to the poor vitality of the cells themselves. It is recommended to use trypan blue staining to calculate cell viability, and cells that reject trypan blue staining should be greater than 95%. If the vitality is low, it is recommended to revive the cells again. Usually, newly revived cells need to be passaged at least 3 times before experiments can be conducted.
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