Flow Cytometry
Flow Cytometry Experimental Control Groups Setting——Single Positive Group
Source: Elabscience®Published: Mar 15,2024
In the multicolor Flow Cytometry experiment, due to the different coverage of the emission wavelength of different fluorochrome, there may be overlap, which will cause certain interference to the data analysis of the experimental results. We usually set a single positive tube to adjust and compensate. At the same time, the single positive tube can also assist us to adjust the channel voltage to prevent the signal from going out of the receiving range.
Here is the emission spectrum of common fluorochrome
A. How to set up single positive tube
The single positive tube is a single dye tube. It represents a sample tube in which only one fluorochrome antibody is added. In general, the experimental panel has several kinds of fluorochrome, and several single positive tubes need to be set. For example: APC, PE, FITC three-color panel, need to set three single positive tube.
B. Requirements for the preparation of a single positive tube
① The sample type and fluorochrome of single positive tube and full dye tube should be consistent;
② The sample treatment of single positive tube and full dyed tube should be the same. For example, if the full dyed tube is treated with cell fixation and permeabilization, the single positive tube needs to be fixed and permeabilized even if it is a surface marker.
③ The voltage of the machine on the single positive tube should be consistent with that of the full dyeing tube;
④ The single tube should ensure that there is sufficient positive cell population.
C. Common problems of single positive control
Question: With the high expression of target marker and obvious differentiation of negative and positive cells, is it necessary to set single positive tubes?
Answer: If the indicators of the panel are less, there is no need to set single positive tubes, and adjust the cell to the horizontal, flat and vertical state by manually adjusting compensation. However, if there are many indicators in the panel, you must set the single positive tubes.
Before compensation
Full dye tube sample results before compensation
After compensation
Full dye tube sample results after compensation
Figure: There is fluorochrome compensation between FITC and PE, but CD4 and CD8 cells are clearly grouped, and the entire panel has only three colors, which can be manually adjusted to compensate.
Question: How to solve the problem that the expression of some markers is low, and the positive group of cells in the single positive tube is not readily discernible?
Answer: When setting the single positive tube, we will find that the positive groups of some low expression markers (Foxp3, IL-17A, IFN-γ, etc.) are very small, and the software can not correctly identify or even can not identify the positive groups of cells when regulating compensation. At this time, we can select some high-expression markers (CD3, CD4, etc.) in the sample, and replace low-expression markers with the same fluorochrome markers.
Attentions
When carry out Flow Cytometry panel design, try to select fluorochrome with less spectral overlap with other selected fluorochromes to reduce compensation.
The compensation value usually does not exceed 100%. If there is more than 100% of the situation, it may be caused by the series dye fracture. In addition, the compensation value will not be negative, if it is, it means that some channels are overcompensated.
After the software automatically adjusts the compensation, you can open the compensation panel and observe whether the compensation of each channel is reasonable. If it is not reasonable, it can be fine-tuned appropriately.
In order to distinguish the negative and positive group more reasonably, we can first screen the corresponding cell group according to FSC and SSC, which is more conducive to regulating compensation.
Above all, it is more convenient to analyze the Flow Cytometry experimental results if the single positive tube is set correctly and reasonably.