A large problem in flow cytometry - isotype control

Antibody Fab segment may appear with the cell surface low affinity, non-specific binding phenomenon, especially when the cell death or cell damage, this non-specific combination will be more obvious. Therefore, the usual experiment we often use the same type of control to determine the background fluorescence level.

We know that the same type of control was once a classical negative reference for flow cytometry, but to achieve the same type of control and antibody cross-reactivity is exactly the same, it requires subtypes, species, fluorescein, fluorescein: protein ratio, concentration and other aspects Indicators can be consistent, for the time being to mention whether they can pick the right, even the manufacturers are not necessarily able to do exactly the same every time, so to find a match with the antibody background color exactly the same type of control is very difficult.

        Therefore, in order to avoid some of the unnecessary errors in setting up the same type of control, we summarize the same type of application, and summed up the circumstances where the need to use the same type of control, and the use of the same type of control need to pay attention to the place. Specific issues are as follows:

1, if the antigen expression is bimodal distribution, and negative and positive peaks were clearly separated, through the distribution of bimodal can understand the experimental results, then the same type of control do not need to set up. As shown below:

2, if you need to detect the cultured cells, then set the same type of control at this time, can only get some information on the ability to reflect the cell adhesion, and it is difficult to reflect the non-specific fluorescence information. It is not ideal to detect the cultured cells and set the same type of control.

3, if the experiment also used a variety of fluorescein, the analysis, found that the effect of fluorescent leakage on the results of the more obvious, and the background fluorescence is not obvious, then it is not appropriate to set the same type of control, in this case the FMO control is the most Good choice. (FMO control refers to the same kind of cells, stained with a fluorescein of all the other fluorescein, so that the various fluorescein can be reflected on the unmarked channel leakage, so that the door can be placed in the appropriate location, so Is the necessary control for the door, and the FMO control is only important when it is necessary to distinguish between positive and negative, and the FMO control is now commonly used in multicolor experiments and is required for multicolor experiments.

4, in the use of the same type of control, found that non-specific binding level is high, may be due to non-specific Fc end and cell binding, resulting in non-specific signal, then we should pay attention to Fc block.

5, to understand the same type of control is only a reference, to help us filter out some of the factors that affect the experiment, but can not rely on this to determine the negative negative demarcation point or set the door, we must know that the same type of control can only reflect the non-specific binding background fluorescence , And background fluorescence, but also by other factors, such as spontaneous fluorescence, high compensation, antibodies, labeling methods, instruments, other reagents or data analysis and other factors will affect the background fluorescence.

6, the need for antibody titration to reduce the non-specific binding background fluorescence, it is not appropriate to set the same type of control. Because the amount of antibody is too much, will cause the negative peak shift and base widening. As shown below:

7, if the test for cell signal transduction and cytokines were tested, not only to set the FMO control, but also need to set a biological negative control, with particular attention to the cells or cells treated with phosphorylation inhibitors.

8, in the multi-color scheme to join the cell reactive dyes, used to remove dead cells. Because non-specific staining of dead cells is strong, if not removed, will lead to higher non-specific binding, affecting the experiment.

9, in addition to the same type of control, according to different circumstances can also set other controls. For example, for unstained cells, determine the background and spontaneous fluorescence of the negative control, set the PMT voltage; completely stained cells, identify some events beyond the axis of the axis, set the PMT voltage; single stained cells / microspheres, adjust the compensation; Or untreated samples, the experimental negative control; positive cells (stimulated or special drug treatment), the experimental positive control.

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