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Technical Column: Frequently Asked Questions and Answers for Flow Cytometry (FCM)
FCM is a technique that uses flow cytometry to perform individual, multi parameter, and rapid qualitative and quantitative analysis or analysis of single row cells or biological particles in a fast linear flow state. However, in the practical application of FCM, obtaining high-quality streaming data is not an easy task. We will now analyze the common problems in FCM experiments, hoping to help relevant experimenters improve the success rate of the experiments.
1、 How to choose a suitable control?
1) Same type control: parallel experiments are conducted using same type antibodies, with the main objective of eliminating non-specific binding between antibodies and samples.
2) Negative cell control: Conduct parallel experiments using known cells that do not express the target antigen, with the main objective of eliminating non-specific binding of antibodies.
3) Secondary antibody control: The secondary antibody is mostly fluorescently labeled with multiple antibodies, and non-specific binding is generally high, which may cause a higher baseline fluorescence value. The main purpose of establishing a secondary antibody control parallel experiment is to eliminate non-specific fluorescence staining of the secondary antibody.
4) Positive control: Generally, cells with known high expression of target antigens are used for parallel experiments, with the main purpose of eliminating factors such as experimental methods, reagents, and operations that may affect the experiment.
5) Blank control: Cells without any labeling, primarily used for measuring basal fluorescence signal expression values.
2、 What is the appropriate amount of cells collected for flow cytometry detection?
When conducting flow cytometry, at least 5000 live cells need to be recorded, and the cell density is generally adjusted to 1 * 106/100ul for flow cytometry.
3、 How to set gating during FCM detection process?
Generally, gates are set based on scattered light, which is commonly referred to as forward scatter (FSC) and side scatter (SSC). The size of FSC is positively correlated with the diameter of cells. Different cells have larger FSC as the cell size increases; Conversely, the smaller. The size of SSC is positively correlated with the quality of intracellular particle structure. Different cells have more complex intracellular particle structures and higher mass, resulting in larger SSC; Conversely, the smaller.
4、 How to adjust compensation during FCM detection process?
In the FCM detection process, if there are multiple colors present, fluorescence compensation adjustment must be performed. To adjust compensation, it is necessary to first understand the situation of double negative cells, and secondly, single positive and double negative cells must be used. Only with negative cells as a reference can compensation be adjusted neither too much nor too little.
5、 Is it necessary to have the same number of cells in each experiment during FCM detection?
When conducting FCM testing, if cell counting is not performed and experiments are conducted based on intuition, it will directly affect the experimental results. When the number of cells is high but the amount of antibodies remains constant, or when the number of cells remains constant but the amount of antibodies decreases, the average distribution of fluorescent antibodies on each positive cell will relatively decrease. It can be seen that different cell counts will affect the final experimental results. Therefore, when preparing samples, it is necessary to perform cell counting to ensure that the number of cells in each group and each experiment remains consistent.
6、 How to perform color matching when FCM detection involves multi-color analysis?
In the FCM detection process, if multi-color analysis is present, although fluorescence compensation can be used to eliminate the influence of fluorescence spectrum overlap. However, excessive adjustment compensation can still affect the accuracy of the measurement to a certain extent. Therefore, we generally recommend choosing fluorescent antibody combinations with less overlap in fluorescence spectra.
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