In flow cytometry, the labeling effect of fluorescent antibodies on single-cell suspensions directly affects the quality of experimental data. Therefore, when purchasing flow antibodies, we need to consider various factors that affect the quality and detection effect of the antibody, such as the specificity of the antibody, the strength of the fluorescein signal, the way of fluorescein labeling, isotype control and so on.
How to choose the right flow antibody?
1. Flow antibody itself is also an antibody, so the selection of flow antibody should first meet the most basic conditions for antibody selection: target protein recognition specificity, reactive species and application experiments;
2. The fluorescent labeling of flow antibody includes direct labeling and indirect labeling. In the process of flow experiment, it follows to minimize the experimental procedure and process to ensure the authenticity and accuracy of the experiment, so it is recommended to use directly labeled antibody for the experiment as much as possible within the scope of conditions;
3. Selection of fluorescent markers: Different fluorescent markers have different intensities on different instruments. Take FACS Calibur instrument for example: PE ＞APC ＞PE-Cyanine 5 ＞PerCP-Cyanine 5.5 ＞FITC ＞PerCP. Generally, PE is the strongest and suitable for weakly expressed antigens, while FITC is the weaker one, which is suitable for strongly expressed antigens and is widely used. FITC is weaker, suitable for strong expression of antigens and is widely used. If a single indicator is detected in the experiment, the user can choose according to the abundance of the target protein expression and take into account the price of the labeled antibody and other factors; if multiple indicators are detected at the same time, the user first needs to confirm how many channels can be detected by the flow cytometer ------- flow antibody can only choose one fluorescein for each channel, and fluoresceins can be freely matched among channels, for example, if the experimenter detects three indicators at the same time, the following chart can be used to determine the number of channels to be detected. For example, if the experimenter detects three indicators at the same time, he/she can choose one appropriate fluorescein marker for each of the three channels of green, yellow and red in the figure below, such as FITC, PE and PE-Cyanine 5. Never choose the fluorescein marker of the same channel for all indicators, in order to prevent overlapping and interfering of fluorescence, which will affect the final results of the experiment. Therefore, the more channels the flow cytometer has, the more surface/intracellular markers can be done simultaneously on the same sample. Commonly used fluorescent markers include FITC, PE, PE-Cyanine 5, PE-Cyanine 5.5, APC and so on.
Selection of isotype control
Flow cytometry is a little different from other antibody-related experiments in that it requires the selection of an isotype control. The isotype control is used to eliminate the background staining caused by the non-specific binding of antibodies to the cell surface, and is equivalent to the negative control of the experiment. The isotype control is an antibody of the same genus, subtype, and fluorescent label as the labeled antibody. For example, for PE-labeled anti-human CD3 antibody, if the antibody is derived from mouse and the subtype is IgG2a, PE-labeled mouse IgG2a must be selected as the isotype control.
Precautions for the use of flow-through antibodies
1. It is recommended that the number of experimental cells and the ratio of antibodies be appropriate. Excessive amount of cells or antibody may affect the results of the experiment, so it is recommended to optimize the experimental conditions before the experiment. Note: The amount of antibody required for different flow techniques varies greatly. For example, conventional flow cytometry (using a sheath-flow system) requires 1×106 cells as the starting sample volume, and the amount of antibody used is 10 μL, while microfluidic flow cytometry requires only 5×105 cells, and the amount of antibody used can be reduced to 5 μL accordingly.
2. Fluorescein-labeled antibodies should be stored at 4°C and protected from light, not frozen.
3. Try to choose the flow antibody which has been verified by experiments to ensure the experimental results, Elabscience® flow antibody has been verified by experiments, and the relevant data charts are provided for users' reference.
Related references
Flow Cytometry (FCM): A technique for the rapid quantitative analysis and sorting of cells or other biological particles (e.g., microspheres, bacteria, small model organisms, etc.) in a liquid stream, one by one, in a single row. Flow cytometry was produced in the 1960s and 1970s, with the advantages of fast speed, high precision and good accuracy, it is a very advanced technique for quantitative cell analysis. It is widely used in cell biology, immunology, hematology, oncology, pharmacology, genetics and clinical testing, such as cell cycle, apoptosis, cell viability detection, the study of the relationship between lymphocyte subpopulations and diseases, immunological monitoring after organ transplantation, detection of tumor-specific indicators, research on the mechanism of action of drugs, and screening of new drugs, and so on.
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