The fact is that once done with accurate staining and Cytometry run, the hard work starts- data analysis. To reliably examine the cells of interest, it is important to assemble knowledge of biology. It needs to do properly, otherwise, the final result will affect. The gating will result in an all-or-nothing data reduction process. The cells available at the gate could drag to the next checkpoint, while the cells available outside the gate will be excluded.
Let’s look at the 4 FMO Control Flow Cytometry gating tips need to be remembered:
- Make sure to check the stability of the run
The best way to check the stability is to Plot a time vs. a scatter plot. It will help you to check the flow during the run. It will also help you to know the flow during the run. It will also help to eliminate artifacts caused by poor flow.
- Take out the doublets
While General Experimental Procedure, try to concentrate on cell clumps which pass through the laser intercept. The fact is that it will take more time than single cells. It also affects the area of the signal. With the use of a pulse geometry gate, the doublets can easily remove.
- Don’t give the top-priority to size
It will be the best to use the scatter gate to eliminate the debris on the left side of the plot. Despite it, also remove the pyknotic or small cells that are mostly FSC small and SSC complex. This effort will help to eliminate both debris and dead cells from your analysis.
- Make sure to consider your control as your guide
The fact is that the controls run in for the experiment are significant to know whether there is an identification of the proper cells. An FMO Control Flow Cytometry, for instance, it is significant to identify the exact placement of a gate in a multicolor experiment. The fact is that the spread of the data because of the fluorochromes in the panel cannot be accurate by using an isotype control. It should be noted that while this process, if the cells in the red circle show cell in the spread region, thereby, it should be excluded. To get entire optimize of this panel, but it would be good to use a brighter antibody.
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Let’s look at the effective recommended for FACS Antibody:Harvest and clean cells and drag cell number to a concentration of 1-5x106 cells/ml in ice cold FACS Buffer.
But you can strain these in any container for which you have the accurate centrifuge such as eppendorf tubes, test tubes, and 96-well round-bottomed micro titer plates.
It is suggested to verify the viability of the cells which should be more than 95% and not less than 90%.
Moreover, it will be effective to do staining with ice cold solutions and at 4°C, because low temperature and presence of sodium azide prevent the modulation and internalization of surface antigens which could produce a loss of fluorescence intensity.Put 100 μl of cell suspension to every tube.The blocking antibody step is required however should be included if cells realizes high levels of Fc receptors.
Now incubate for at least half an hour at room temperature or 4°C in the dark.Clean the cells 3-4 times by centrifugation at 1500 rpm for ten minutes and then resuspend them in 200 μl to 1ml of ice cold FACS buffer.
Then clean the cells at least 5 times with the aid of centrifugation at 1500 rpm for 10 minutes and resuspend them in 200 μl to 1ml of ice-cold FACS buffer.
