GUIDELINE
- It is to add fluorescent monoclonal antibodies for specific molecules into a group of mixed cells. This specific monoclonal antibody combines with its corresponding antigenic target molecule to become the target cell labeled by the fluorescent antibody.
- When each cell is irradiated by the laser beam of the instrument, the fluorescence on the cell will be activated by the corresponding laser beam and emit the corresponding fluorescence. Fluorescence from the cell surface can be detected by a sensitive photomultiplier tube.
- In a flow cytometry separation device, different cell subpopulations are labeled with different fluorescent antibodies and carry different physical (particle size, density, fluorescence intensity) information to separate target cells from the mixed cell population.
METHODS
- Vortex the samples before placing the tubes into the FACS instrument. To analyze and sort the cells, adjust the gates in the forward scatter-area (FSC-A) and the side scatter-area (SSC-A) to exclude cell debris and include cells of interest.
- Set the gates in FSC-A and forward scatter-width (FSC-W) to exclude cell aggregates.
- Analyze the following control samples. Tube 3 (WT cells unstained as a control for transgenic hGFAP-eGFP cells and EGFR and PI cells); tube 4 (cells stained with PI to analyze the rate of cell death); and tube 2 (rat IgG1 K isotype control PE, as an isotype control for prominin1 cells).
- Determine the rate of cell death by measuring the proportion of PI cells using tube 4. Discard all experiments with cell death rates higher than 5%.
- Set the gates for hGFAP-eGFP and the EGF receptor ligand conjugated to Alexa Fluor 647 by using WT unstained cells (tube 3) and the isotype-matched antibody control conjugated to PE for prominin1-PE (tube 2).
- Then, using tube 1, sort the quiescent NSCs (hGFAP-eGFP, prominin1, EGFR++−), activated NSCs (hGFAP-eGFP, prominin1, EGFR), niche astrocytes (hGFAP-eGFP only) and ependymal cells (prominin1 only) simultaneously.
- Collect the cells directly into FACS tubes suitable for sorting (nonadherent or coated), containing 1-2 mL of culture medium or buffers, depending on further applications.
- The identity of the sorted cells is examined by inoculating the cells onto a poly-lysine-coated cover slide, followed by fixation and immunocytochemistry.
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NOTES
- When the voltage is adjusted, if the volume of the detected target cells is small, the voltage value can be increased appropriately, so that the target cells and cell fragments can be completely separated in flow cytometry. If the volume of the detected target cells is large, the voltage value can be lowered appropriately so that all the target cell populations can be completely displayed in the flow diagram, to prevent the cell populations from being deformed and distorted close to the boundary of the flow diagram or completely outside the boundary.
- Indirect labeling of secondary antibodies will increase the experimental steps, cause cell waste after repeated cleaning, and affect the accuracy of the experiment. Therefore, it is suggested to try to use directly labeled antibodies for the experiment.
- Strong fluorescence is recommended for weak antigen expression or not obvious grouping, and weak fluorescence is recommended for vice versa.
- Only one luciferin can be selected for each detection channel. (For example, cells with GFP can no longer be stained with FITC channel antibodies, which will overlap signals).
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