Hi user, No. Those Fluorescent Minus One (FMO) controls do not contain any isotype antibodies. Those FMOs contain all of the stains used in the experiment except for the stain referenced in the FMO, ie the IFN FMO has all of the stains except for the IFN stain. We hope this helps!

Hi user, After setting the axis to linear, draw an initial gate around the G1 peak. Then set constraints to generate the rest of the model. I almost always draw a gate on the G1 peak, then set G2 = G1x2 and set the G2 CV = G1 CV. The G2 peak should theoretically have about twice the intensity of the G1 peak (though sometimes it has slightly less). The CV of the G1 and G2 peaks should theoretically be the same. If you need further assistance please reach out at [email protected]!

Hi user, as long as the peak is still within the G1 gate, the model should work as expected. If the peak has shifted outside of the gate then the G1 gate will need to be shifted to include the G1 peak. If you have more questions please email us at [email protected]!

@@FlowJoMediaHi, the suggestion of shifting the gate is contrary to what Jack indicated in the video. The essence of gating is to be able to use the control as a standard to compare with the other tests.

Hi, Different experiments will use different dyes to calculate ratio parameters describing the change in cell expression. We have more information on creating derived parameters in our documentation. Also, feel free to reach out to [email protected] to discuss your experiment. docs.flowjo.com/flowjo/experiment-based-platforms/plat-derived-overview/

Hi, For the data Jack presented the cells were released from an inhibitor keeping them in the G1 phase before the experiment began. Because the data was acquired shortly after the cells were released from the G1 phase, most of the cells for the experimental samples were in S phase. The distribution of cells in other cell cycle experiments may look different depending on the inhibitor used and the time at which the data was acquired.

@@FlowJoMedia Many thanks. In addition, it seems single cell discrimination was not carried out after gating out cells from debris.

Hi user, The performance may be boosted if the performance preferences are edited. These preferences can be found under the heart shaped icon and then choosing “Performance”. For Windows users the “Max Sample Cache Size” may be lower than optimal. If many gigabytes of data are being loaded into the workspace a higher sample cache size may be more optimal. Here are some suggested sample cache sizes: For 8 GB of RAM ~2000 For 16 GB of RAM ~8000 For 32 GB of RAM ~16000 For 64 GB of RAM ~32000 Issues can occur when the .wsp or .fcs files being used in the workspace are stored on a server. This is because the computer can often have poor or inconsistent connection to files stored in remote locations. If the files are stored on a server, I would try moving them locally (ie to the desktop) and see if that helps. Below is a link to more information on using FlowJo with a remote drive. docs.flowjo.com/flowjo/advanced-features/remote-data/

Hello, please reach out to [email protected] and we will be happy to assist you with this issue!

Hi Felipe, please reach out to us at [email protected] and we can assist you further and discuss some possibilities in this case. Thank you!

Please reach out to [email protected] and we will be happy to assist you!

Glad to hear that!

Glad it was helpful!

Hi Angela, Yes. Gates can be made before or after tSNE. Hope this helps!

Hi Jacqueline, Autospill compensation requires no negative control. An unstained cell control can optionally be added to an empty detector in order to estimate the background fluorescence present in the cells. Then when compensation is performed, the signal from the unstained control present in each other detector will be removed, subtracting out the autofluorescent signal. Learn more about Autospill compensation on our website: docs.flowjo.com/flowjo/experiment-based-platforms/plat-comp-overview/autospill-compensation/

Hi Vincent! Based on our testing (presented in this video), using option 1 (acquire with all PMTs opened and use spectral compensation) will give you less spread of your data than option 2 (using non spectral comp). So I you have spreading issue with your data that cannot be resolved with better panel design, I will recommend you to acquire your data with all PMTs opened and use spectral compensation. Best

@@NicolasLoof Dear Nicolas, thanks for your reply.

Hi Faisal, please reach out to at [email protected] for more support here!

Hi Jennifer, Some Cytometers do not display record a time parameter. FlowJo does have a Event Number parameter that can function similar to time, displaying the sequence the events were recorded into the file by the Cytometer. This can be found under the preference “Heart icon” in the “Graphs” section. Hope this helps!

Hi Barbara! Please reach out to [email protected] for assistance with this and we'll be happy to help.

Hi, unfortunately, you cannot derive a p value in FlowJo. You must export your data using the Table Editor and derive the p value using Excel, Prism or some other software.