Steps involved in compensation by FlowJo: It is important to remember that the negative population and positive population must share the same autofluorescence characteristics for compensation to be valid (i.e., don't use monocytes as a negative control for a lymphocyte stain if you are compensating a stain on fibroblasts, use an unstained fibroblast control). Ideally, you would use a stain that only labels a portion of the sample population, so that you have an unstained set of cells in each tube. Thus, you must collect samples that are singly-stained (as well as unstained) for each of the fluorescences that require compensation. To do this, you will select gates on positive and negative populations for each of these stain, and tell FlowJo to calculate the compensation matrix based on these stains. The remainder of the documentation below is NOT meant to teach you about compensation, simply how to use FlowJo to perform that function.įlowJo computes the compensation matrix on control samples much the way you would manually set the compensation. Also, there may be cases where the instrument is not capable of compensating between certain channels (for instance, to correct for the spillover between fluorescein and Cy5PE).įor a description of why compensation is necessary, the underlying concepts behind compensation, and some of the pitfalls of improper compensation, see the " Compensation: A perspective" by Mario Roederer (you need to be connected to a network to view this site). This may be necessary in cases where the compensation was inappropriately set during sample collection (although if the sample was over-compensated, then there is no recourse). we acquire our samples on BD LSRII.FlowJo gives you the ability to compensate your data. > I have a small query about compensation in On Jan 21, 2010, at 5:54 AM, Rashim pal Singh wrote: You can edit your workspace columns toĭisplay the compensation matrix name that is applied to your sample. As pointed out, if you want, you can use the singleĬolor controls to calculate a matrix in Flowjo - it should be the sameĪs what is calculated by DiVa. If you tell FlowJo toĭisplay uncompensated parameters, you can view your data compensated Your samples and call it the Acquisition Matrix. When you export your data out of DiVa and load it into FlowJo, theĬomp matrix is part of the FCS header, and FlowJo iwill apply it to Use calculate and use the proper compensation matrix with each sample. Reagents conjugated to PE-Cy5, you can set up the DiVa compensation to Tandem fluorchrome in your experiment, for example two different If you are using two or more different Abs conjugated to a particular Panel to display compensated or uncompensated data. You can turnĬompensation on or off using the Compensation tab in the Cytometer The DiVa software will allow you, using single color controls, toĬalculate a compensation matrix for your experiment. The data collected using the DiVa software is always uncompensated. To older versions of Diva (v4.x), as I haven't experienced this with theįrom: cytometry-bounces at Through some variable testing, I determined that Diva export was most Or, just export uncompensated data from Diva and generate all Then to import the data into FlowJo and strip off the original comp One way around this was to make sure the comps were correct in Diva, ![]() Were at zero (as if no comps had been applied). Was no way to correct it in FlowJo as all of the spillover corrections Result of this, if the original data were at all overcompensated, there Restored the original compensated data from Diva. The problem was that the dataĬompensated in Diva prior to export were being treated as uncompensatedĭata when imported into FlowJo, so the comp matrices attached to theįCS3 files were essentially "re-applied" on top of the existing ones, I used to have problems adjusting compensations after exporting FCS3ĭata from Diva and importing into FlowJo. The latest version of DIVA would be best,but that is another story.įrom: cytometry-bounces at On Behalf Of Collin Spencer Make sure you have the latest version of FJ. If not satisfied then email Adam himself. I have always gotten prompt answers from them. ![]() I suggest we let TreeStar answer the original and always suggest you email TreeStar before you ask this from the list-serv.
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