Multi-parameter analysis of apoptosis using flow and image cytometry
Multi-parameter analysis of apoptosis using flow and image cytometry
Apoptosis regulates the dynamic balance of cells, and the multiparametric nature of flow cytometry allows for the simultaneous determination of several apoptotic indicators in the same specimen, providing a powerful tool for analyzing the complex apoptotic process of different cells. Currently, flow and image cytometry are used to analyze apoptosis.
Principle
The basic principle of multiparametric analysis of apoptosis by flow and image cytometry is to utilize DNA-binding dyes as membrane permeability indicators (e.g., propidium iodide, PI) in combination with other indicators to evaluate different cellular responses related to apoptosis, including e.g., mitochondrial membrane potential, annexin V that binds to "flipped" PS, etc., providing a broader and more multidimensional picture of the entire cell death process. This provides a broader and multidimensional picture of the whole cell death process.
Operation method
Multi-parameter analysis of apoptosis using flow and image cytometry
Principle
The basic principle of multiparametric analysis of apoptosis by flow and image cytometry is to utilize DNA-binding dyes as membrane permeability indicators (e.g., propidium iodide, PI) in combination with other indicators to evaluate different cellular responses related to apoptosis, including e.g., mitochondrial membrane potential, annexin V that binds to "flipped" PS, etc., providing a broader and more multidimensional picture of the entire cell death process. This provides a broader and multidimensional picture of the whole cell death process.
Materials and Instruments
Equipment: Flow cytometer. Move The experimental steps for assessing lymphocyte-mediated cytotoxicity using flow cytometry were as follows: A. Collect suspended cell lines or cultured primary cells and transfer to 12 x 75 mm cell culture tubes. B. Centrifuge the cells at 400 g for 5 min and discard the supernatant. Removal of the supernatant is critical for the following steps and the amount of supernatant left should be as small as possible. C. Label (0.5-2) × 106 cells per sample; increasing the amount of cells will decrease the efficiency of caspase and annexin V labeling.
Reagent: PhiPhiLux-G1D2 caspase 3 fluorescent substrate.
A. Cells are first labeled with PhiPhiLux caspase substrate.
B. Tap the tube and resuspend the cells in the residual supernatant to a volume of approximately 50ul of supernatant in the tube.
C. Add 50ul of PhiPhiLux Reagent to each tube and shake gently. (See Note 4.4).
D. Incubate at 37°C for 45 min.
(iii) Labeling of Annexin VA. Cells are labeled with fluorescent dye coupled to annexin V.
B. After incubation for 45 min, remove supernatant from the above tubes and add appropriate fluorescent dye-coupled annexin V (PE or APC).
C. Incubate at room temperature for 15 min.
D. Add 3 mL of wash solution to each tube, centrifuge at 400 g for 5 min, and gently decant the supernatant.
(iv) DNA Binding Dye LabelingA. Dissolve PI (2pLg/mL) or 7-AAD (5jjig/mL) in complete medium.
B. Add 0.5 mL of PI or 7-AAD solution to the tubes described above after removal of supernatant. Samples should be left at room temperature and analyzed within 60 min.
(v) Flow cytometry analysisCells should be analyzed as soon as possible to minimize apoptosis after the experiment. The instrument should be set up and sample collection completed immediately. Fluorescence detector assignment and analysis problems:
A. PhiPhiLux-G1D2: A fluorescent yellow-like caspase substrate that is analyzed on most flow cytometers through the fluorescent yellow or FITC channel (defined as FL1), often paired with a 530/30 nm or similarly narrow band-pass filter The spectral characteristics of PhiPhiLux-G1D2 fluorescein are similar to fluorescent yellow. Adjustment of color compensation is required for simultaneous use with PE or PI (rarely used with 7-AAD).
B. PE-COUPLING ASSOCIATED ASSOCIATED V: Most PE- coupled reagents, detected through the PE channel (usually defined as FI2), are often coupled with a 575/26-nm or similar band-pass filter. When used with PhiPhiLux-G1D2 and 7-AAD, color compensation needs to be adjusted.
C. APC coupled annexin V: Excited by a red laser and detected on many flow cytometers through the APC channel (often defined as FI4) with a 660/20nm or similar bandpass filter, the great advantage of APC in multicolor analysis is that it requires very little adjustment for compensation and there is no significant spectral overlap with PhiPhiLux-G1D2, PI, or 7-AAD.
D.PI: This DNA binding dye is very bright even at very low concentrations, has a wide emission range, and requires color compensation when used with PhiPhiLux-G1D2. It can be detected in the PE or far-infrared detection channel (with PE575/26nm filters or the longer red spectrum), while the latter reduces fluorescence leakage into the fluorescein detector.
E.7-AAD: This DNA-binding dye is darker than PI, emits far-infrared light, and can be detected in the far-infrared channel of most flow cytometers (defined as FL3) with a 675/20 bandpass or 650nm dual-color or similar filter. Color compensation is required for use with PhiPhiLux-G1D2 and PE. In some multicolor measurement systems, it is not known why 7-AAD slightly bursts PE, so this should be considered when installing the instrument.
(vi) GatingA. Scattering Gates: Most cell lines and some primary cells will show large changes in forward or side scatter analysis during late apoptosis (cell size or optical density are rough indicators). Therefore, it has been suggested that a population of live scattering cells should be captured to observe caspase activity, annexin V binding, and DNA dye uptake alone as a measure of early cell death. However, the scatter apoptotic cell population may contain some live cells that undergo transient changes in cell volume prior to other markers of cell death such as caspase activation. Therefore, to accurately analyze cell death, the entire population of scattered cells should be selected (excluding obvious fragments). Subsequent gating of live cell populations (which may actually contain early apoptotic cells) should be performed on the basis of annexin V or DNA dye binding.
B. Exclusion of annexin V binding and DNA binding dyes: These markers often occur after caspase activation and are considered late apoptotic markers. Therefore, positive and negative subpopulations of annexin V and DNA-conjugated dyes can be used to set up gates to distinguish between early and late apoptotic cells. Subsequent caspase activation analysis of these cell populations can further distinguish the early stages of apoptosis.
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