FlowJo is the leading platform for single-cell flow cytometry analysis. We’re here to help you accelerate routine phenotyping, take your immunology research to the next level, and get you from data to results―one cell at a time.
Do all samples have to follow the same constaint n value that was done with a negative control or are we allowed to tweak n values per sample to minimize RMSDs?
Thank you, FlowJo. As a further clarification, I think gating the phases for the control for use with other samples should only be applicable if the cells were synchronized when the treatments were carried out. Could you kindly confirm?
Very usefull video. Would´ve been nice to watch the whole procedure, from FSC/SSC --> FL2A/FL2W(or other doublet discrimination method) --> Cell cycle analysis
Hi Serena, in order to use CytoNorm the reference control must be stained with the same panel of the batch samples (same fluorochromes or number of marker etc)? Thank you!
Presenter could briefly pause in the drop menus before he clicks on them so that the viewer doesn’t have to go back in the clicks several times to pause at the precise instant he quickly clicked on.
Thank you for your tuto. I have a question about your FMOs used to determine the positive gates for IFN, Perf etc. Have they been done with isotype antibodies? Thank you
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 FlowJo@bd.com!
what happens if the position of G1 shifts between samples and I applied the same model to every sample (restricting G1): For instance, the peak appears in 200 in one sample and in 230 in another sample. I should manually reset the position of said sample, shouldnt I?
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 FlowJo@bd.com!
@@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.
if you guys are going to use shortcuts, you should write them on the video. This should also just be a written tutorial so people don't need to watch videos to use a product
Dear Jack, my name is Antonia and im currently working on a calcium influx study, but I am having troubles using the kinetics tool in the latest version of FlowJo v10. I think my problem ist, that im not able to set the right derived parameter to show the kinetic curve of stimulated platelets. Can you explain what parameter you are using here. I can see you named it Ratio: DAPI_A_Ido-1. What setting is behind that parameter? Thank you in advance for helping!
Hi sir: At minute 21:20, you made a statement which is contrary to the data. More cells in G1 phase for the control, but it's not so. How will your clarify this, please?
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.
Amazing Tutorial, Thank you so much! I have a question about performance of FlowJo. I got Problems when applying gates to other samples. My FlowJo crashes or goes in a state of infinite loading time. I already set the memory use to 1/4of my RAM. Any suggestions? thanks again! :)
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 again, here at ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-5_AB-qVEZqs.html, for Positive, you use -for ex- "Size/APC-H7-A+", so you need to adjust the "APC-H7-A+" gate or is it ok to use "only" as positive the "Size" (or Clean up) gate? Thanks
Hi! Thank you for the talk. In this video you created categorical gates on a concatenated file first and then did TSNE. Is it OK to run TSNE first & then add the categorical gates? Thanks.
In autospill I saw that you removed your universal negative after adding an unstained control to an empty channel. do you have to re-assign that sample as the universal negative? If not, then how does compensation get subtracted/what would be the purpose of adding it to an empty channel? Thanks so much for this tutorial! It was super helpful.
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/
Nicolas, many thanks for this webinar. One question: when using a 10 colors "traditional" instrument, and running a 5colors panel, is it better to 1) acquire with all PMTs opened and use spectral compensation or 2) close unused PMTs and go for traditional or Autospill compensation? Thanks, Vincent
Hi, just want to inquire that how I should manage the CFSE stained unstimulated control for the undivided population? should I use the time zero analysis for this control on the time zero or should I wait for the 72 hrs.??
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!
