Yes I always do. I would recommend at the very least test it on your system and see what the compensation values are. Sometimes they are very low so you may be able to reuse the compensation matrix.
@@job506 the high FSC events? I excluded these because they tend to be clumps/doublets. You could include these and then use doublet gating to exclude them, if you wanted.
Hi Aja, sorry if I am asking basic question. Could you advise some tips to set up the control cells for compensation purpose? Our cells after compensated doesn’t have such beautiful shapes like yours. Thank you!
Not basic at all- this is a critical question! What works best really comes down to your cells. Apoptosis can be induced via ultraviolet B irradiation, small molecule drug treatments, death receptor ligation, and exposure to granule components of cytotoxic lymphocytes. I have generally used cholchicine when using small molecules. Heat shock can work well for some cells. Or you can always fix the cells (with formaldehyde or alcohol)- this will result in a strong positive stain but you'll be left with no negative... so if using fixative, add some unfixed live cells back in so you have both populations. I hope this helps!
Hi aja. I did Annexin-V/FITC staining. I induce apoptosis in one of my sample. In another samples i treated cells with my drug in the presence of positive control. I can see the difference that drug is preventing the cells from undergoing apoptosi. Now i dont know how to calculate the apoptosis cells ratio? Will you help me with this plz?
Hi! Sorry I've gotten behind on my replies. Generally in this case people will report %+ for AnxV+/PI-, AnxV+/PI+, AnxV-/PI+. If you are only interested in one of those populations then you would simply report that.
First and foremost, I want to extend my gratitude for the incredibly informative tutorial video! Thank you for sharing your knowledge and expertise through this fantastic video!! After watching your tutorial, I have been trying to apply the analysis techniques to my apoptosis data. However, I am facing some challenges. Specifically, I am encountering issues with signal saturation during the analysis, even when I set the detector voltages to low values. I am currently using a commercial kit, the FITC Annexin V Apoptosis Detection Kit, and carefully following the manufacturer's staining protocol, but the problem persists... I am seeking your expertise to help identify the root cause of this issue. If there are any specific parameters, settings, or alternative approaches that I should consider, I would greatly appreciate your input~
Sorry I missed this one! I would recommend titrating down your reagents so that you can reduce the signal saturation. I have found most of these kits use a way higher concentration than is actually needed.
It looks like my comment was deleted so I'll try again (Might be because I posted an imgur link). First, thanks for the informative videos! I'm very new to flow cytometry and am trying to do some cell cycle and apoptosis analysis. After acquiring my data in .fcs file I proceeded to follow the instructions in your videos. However, my X and Y-axis options do not have the usual FSC, SSC, FITC, PI, etc etc. Instead, I have options like "Area", "Aspect ratio", "Gradient RMS", "Background mean" etc etc. Any help or solutions? My instrument is Amnis ImageSteam X MkII, if that helps.
Glad this went through! It’s the first time I’ve seen it. Having acquired your experiment on the ImageStream, I would really highly recommend analyzing it in IDEAS (the ImageStream software) rather than as an exported FCS in FlowJo. You get such a wealth of cell death information from the images, it would be a shame to lose out on that! IDEAS has a great analysis wizard that you can use to help out and reduce the learning curve for the software (it can be quite steep). If you do want to use FlowJo, the most similar axis labels would be: FSC = Area M01 (assuming this is your bright field channel) SSC = Intensity Ch 6 or 12 (depending on your configuration and how the data was acquired) AnxV = Intensity Ch 2 (assuming from your comment that you are using FITC) PI = Intensity Ch 4 All that being said- do the analysis in IDEAS! You will get so much more information out of it!
@@ajarieger_flow Thank you so much for the reply! You are such a lifesaver. I am aware of IDEAS, but the technical officer that provided me with some training with ImageStream told me IDEAS is a difficult software to use so I ended up looking for alternatives. Of which I stumbled across your channel and FlowJo seemed simple and intuitive enough so I went with FlowJo. Given your advise I'll definitely go back to IDEAS and see what I can achieve there. Hilariously enough even though my uni has a pretty advanced flow cytometer (ImageStream), very little people, even the technical officer herself, knows how to really operate the instrument to its full potential. The only unfortunate thing is that I acquired my data only in .fcs format, and not in .rif that IDEAS recognize. I tried importing my .fcs into IDEAS earlier and nothing really happened. I'll have to run my experiments again. But that's alright since its part of the learning process. In the meantime I'll mess around with FlowJo first with the help that you've provided. Again thank you so much for the help. Really really really appreciate your time. I hope you have a nice weekend!
@@ajarieger_flow Oh my god I think YT deleted my comment AGAIN. I didn't include any links this time. YT please D: Anyway, thank you so so much for your help! You are such a lifesaver. I am aware of IDEAS. However, the technical officer who gave me some training to operate ImageStream told me the software is difficult to use (She doesn't even know how to use it herself). So I opted for alternatives. Of which I stumbled across your channel, and FlowJo seemed simple and intuitive enough so I decided on FlowJo. After reading your advise I'll definitely give IDEAS another go. Hilariously enough, even though my uni has a pretty advanced flow cytometer (The ImageStream), very few people, including the technical officer herself, know how to operate the instrument to its full potential. The only unfortunate thing is that I only acquired my data in .fcs format, and not in the .rif format that IDEAS mainly recognize. I tried to import my .fcs data into IDEAS but nothing really happened (Sometimes the software even crashes lmao). I'll have to run my experiments again but its not a big deal because I just did a trial run to get familiar with the process. In the meantime I'll mess around with FlowJo first. Again thank you so much for your help! I really appreciate your time and effort. Hope you have a nice weekend!
@ajarieger_flow Oh my god I think YT deleted my comment AGAIN. I didn't include any links this time. YT please D: Anyway, thank you so so much for your help! You are such a lifesaver. I am aware of IDEAS. However, the technical officer who gave me some training to operate ImageStream told me the software is difficult to use (She doesn't even know how to use it herself). So I opted for alternatives. Of which I stumbled across your channel, and FlowJo seemed simple and intuitive enough so I decided on FlowJo. After reading your advise I'll definitely give IDEAS another go. Hilariously enough, even though my uni has a pretty advanced flow cytometer (The ImageStream), very few people, including the technical officer herself, know how to operate the instrument to its full potential. The only unfortunate thing is that I only acquired my data in .fcs format, and not in the .rif format that IDEAS mainly recognize. I tried to import my .fcs data into IDEAS but nothing really happened (Sometimes the software even crashes lmao). I'll have to run my experiments again but its not a big deal because I just did a trial run to get familiar with the process. In the meantime I'll mess around with FlowJo first. Again thank you so much for your help! I really appreciate your time and effort. Hope you have a nice weekend!
Hi Aja, I am having difficulty compensating my annexin-FITC stained cells.FlowJo is new for me. I have used FACSDiva a few times, Your samples look great. Mine seem uncompensated, and I watched so many UA-cam videos for compensation, however, it does not work. I am not able to see the Annexinv-FITC-A parameter, I have FITC only, also No PI, only PE. I have done Annexin-V apoptosis with 16 different samples: - unstained (no drug treatment), annexin v-FITC only (no drug treatment) , PI only ( no drug treament ), PI and annexin-vFITC (no drug teatment ). -unstained (250 drug treatment), annexin v-FITC only (250 drug treatment), PI only (250 drug treatment), annexin v-FITC and PI (250 drug treatment) -unstained (125 drug treatment), annexin v-FITC only (125 drug treatment), PI only (125 drug treatment), annexin v-FITC and PI (125 drug treatment) -unstained (H202 treatment), annexin v-FITC only (H202 treatment), PI only (H202 treatment), annexin v-FITC and PI (H202 treatment) ONLY the first 4 are for compensation ?? I am not sure how to analyze the data. Could you please lease please help ? Can I please send the FCS files to your email please?
Thank you so much for all the informative videos on FlowJO. I have a question I am trying to check the mitochondrial membrane potential of cells for which I am using JC-1 stain. It will tell about two populations. How to do an analysis for that? Which filter to use? Do I need to do the analysis in FL1-FITC and FL2 PE-A? I am confused. Kindly help.
Happy to help! JC-1 will be "red" when the mitochondria are active and "green" as they lose membrane potential. Both will be excited off at 488nm laser on most flow cytometers. The RED fluorescences at 590nm; the GREEN at 529nm. Not knowing the setup of your instrument, I don't know channels they will be in. But look at the setup and see which channel will capture this emission. Hope that helps!
@@ajarieger_flow Thank you so much. I have already acquired the data. I want to know how to analyze the data and differentiate bw two different populations with HMMP and LMMP. Thank you so much in advance.
Hi Aja, I am doing my first-time flow cytometry analysis using flowjo. But I am not able to see the BL1-A ::Annexinv-FITC-A option in my screen. instead of that I AM seeing CoMP-FL1-A :: FL1 INT LOG , this option. Also i have a doubt, I have done flow cytometry with 8 samples..... unstained( no drug treatment ), annexin v only ( no drug treatment ) , PI only ( no drug treament ), PI and annexin v (no drug teatment ). unstained ( with drug treatment), annexin v only ( with drug treatment ), PI only ( with drug treatment), annexin v and PI ( with drug treatment) So here first 4 are for compensation ?? I am not sure how to analyze the data. could you please help to give some idea.
Hi! Without seeing the data, it is hard to say for sure. However, this indicates to me that you compensated when you acquired the samples and that’s why they show up as “COMP…”.
Thanks for the quick response. As this is my first time with flow cytometry I am having a lot of trouble with the analysis. IF possible can I share you my data?
![Adjusting compensation: how to handle a "bad" matrix [FlowJo advanced]](http://i.ytimg.com/vi/NEtDeck_V3I/mqdefault.jpg)
![Adjusting compensation: how to handle a "bad" matrix [FlowJo advanced]](/img/tr.png)
![FlowJo [tSNE]](http://i.ytimg.com/vi/UK725D14-bo/mqdefault.jpg)
I watch this video every time I calculate apoptosis. I have it bookmarked! thank you so much for this video!
I’m glad it’s helpful! Let me know if there’s anything else that would be useful for you 😊
Thank you a lot for this informative a straightforward video!
add how to calculate and apply compensation for annexin-PI before doing this analysis. It will be a great help
What an amazing and simple video! Thanks a lot!!
If there’s a topic you’d like a video on, just let me know!
Thanks for this amazing content!!!
Thanks for your explanation!
Did you carry out any compensation for both FITC and PI, please?
Yes I always do. I would recommend at the very least test it on your system and see what the compensation values are. Sometimes they are very low so you may be able to reuse the compensation matrix.
@@ajarieger_flow Many thanks. It seems you already compensated at the time of data collection rather than during data processing with FlowJo.
@@job506 you can either way, depending on if you did compensation during the acquisition or afterwards in FlowJo.
Hi, I really enjoy your videos. Hope you can make a video about NETs on flow cytometry
Thanks!! I’ll add it to my list 😊
@@ajarieger_flow also a CBA video would be interesting and easier than NETs 😅
Multiple thanks for the video. Please, how do you account for the fact that some of the cells were on the chart edge?
Happy to help! Are you talking about events on the chart edge in the negative area or the positive area (off-scale)?
@@ajarieger_flow ..events that were excluded in the positive area during gating @ 0.59 second.
@@job506 the high FSC events? I excluded these because they tend to be clumps/doublets. You could include these and then use doublet gating to exclude them, if you wanted.
Hi Aja, sorry if I am asking basic question. Could you advise some tips to set up the control cells for compensation purpose? Our cells after compensated doesn’t have such beautiful shapes like yours. Thank you!
Not basic at all- this is a critical question! What works best really comes down to your cells. Apoptosis can be induced via ultraviolet B irradiation, small molecule drug treatments, death receptor ligation, and exposure to granule components of cytotoxic lymphocytes. I have generally used cholchicine when using small molecules. Heat shock can work well for some cells. Or you can always fix the cells (with formaldehyde or alcohol)- this will result in a strong positive stain but you'll be left with no negative... so if using fixative, add some unfixed live cells back in so you have both populations. I hope this helps!
Hi aja. I did Annexin-V/FITC staining. I induce apoptosis in one of my sample. In another samples i treated cells with my drug in the presence of positive control. I can see the difference that drug is preventing the cells from undergoing apoptosi. Now i dont know how to calculate the apoptosis cells ratio? Will you help me with this plz?
Hi! Sorry I've gotten behind on my replies. Generally in this case people will report %+ for AnxV+/PI-, AnxV+/PI+, AnxV-/PI+. If you are only interested in one of those populations then you would simply report that.
First and foremost, I want to extend my gratitude for the incredibly informative tutorial video! Thank you for sharing your knowledge and expertise through this fantastic video!! After watching your tutorial, I have been trying to apply the analysis techniques to my apoptosis data. However, I am facing some challenges. Specifically, I am encountering issues with signal saturation during the analysis, even when I set the detector voltages to low values. I am currently using a commercial kit, the FITC Annexin V Apoptosis Detection Kit, and carefully following the manufacturer's staining protocol, but the problem persists... I am seeking your expertise to help identify the root cause of this issue. If there are any specific parameters, settings, or alternative approaches that I should consider, I would greatly appreciate your input~
Sorry I missed this one! I would recommend titrating down your reagents so that you can reduce the signal saturation. I have found most of these kits use a way higher concentration than is actually needed.
In addition, how many cellular events did you consider to get this dot plot?
I got this already. thanks.
Great! Just so we're on the same page, I generally will record a minimum of 10,000 single cells for AnxV/PI analysis
It looks like my comment was deleted so I'll try again (Might be because I posted an imgur link).
First, thanks for the informative videos! I'm very new to flow cytometry and am trying to do some cell cycle and apoptosis analysis. After acquiring my data in .fcs file I proceeded to follow the instructions in your videos. However, my X and Y-axis options do not have the usual FSC, SSC, FITC, PI, etc etc. Instead, I have options like "Area", "Aspect ratio", "Gradient RMS", "Background mean" etc etc. Any help or solutions? My instrument is Amnis ImageSteam X MkII, if that helps.
Glad this went through! It’s the first time I’ve seen it.
Having acquired your experiment on the ImageStream, I would really highly recommend analyzing it in IDEAS (the ImageStream software) rather than as an exported FCS in FlowJo. You get such a wealth of cell death information from the images, it would be a shame to lose out on that!
IDEAS has a great analysis wizard that you can use to help out and reduce the learning curve for the software (it can be quite steep). If you do want to use FlowJo, the most similar axis labels would be:
FSC = Area M01 (assuming this is your bright field channel)
SSC = Intensity Ch 6 or 12 (depending on your configuration and how the data was acquired)
AnxV = Intensity Ch 2 (assuming from your comment that you are using FITC)
PI = Intensity Ch 4
All that being said- do the analysis in IDEAS! You will get so much more information out of it!
@@ajarieger_flow Thank you so much for the reply! You are such a lifesaver.
I am aware of IDEAS, but the technical officer that provided me with some training with ImageStream told me IDEAS is a difficult software to use so I ended up looking for alternatives. Of which I stumbled across your channel and FlowJo seemed simple and intuitive enough so I went with FlowJo. Given your advise I'll definitely go back to IDEAS and see what I can achieve there. Hilariously enough even though my uni has a pretty advanced flow cytometer (ImageStream), very little people, even the technical officer herself, knows how to really operate the instrument to its full potential.
The only unfortunate thing is that I acquired my data only in .fcs format, and not in .rif that IDEAS recognize. I tried importing my .fcs into IDEAS earlier and nothing really happened. I'll have to run my experiments again. But that's alright since its part of the learning process. In the meantime I'll mess around with FlowJo first with the help that you've provided.
Again thank you so much for the help. Really really really appreciate your time. I hope you have a nice weekend!
@@ajarieger_flow Oh my god I think YT deleted my comment AGAIN. I didn't include any links this time. YT please D:
Anyway, thank you so so much for your help! You are such a lifesaver.
I am aware of IDEAS. However, the technical officer who gave me some training to operate ImageStream told me the software is difficult to use (She doesn't even know how to use it herself). So I opted for alternatives. Of which I stumbled across your channel, and FlowJo seemed simple and intuitive enough so I decided on FlowJo. After reading your advise I'll definitely give IDEAS another go. Hilariously enough, even though my uni has a pretty advanced flow cytometer (The ImageStream), very few people, including the technical officer herself, know how to operate the instrument to its full potential.
The only unfortunate thing is that I only acquired my data in .fcs format, and not in the .rif format that IDEAS mainly recognize. I tried to import my .fcs data into IDEAS but nothing really happened (Sometimes the software even crashes lmao). I'll have to run my experiments again but its not a big deal because I just did a trial run to get familiar with the process. In the meantime I'll mess around with FlowJo first.
Again thank you so much for your help! I really appreciate your time and effort. Hope you have a nice weekend!
@ajarieger_flow Oh my god I think YT deleted my comment AGAIN. I didn't include any links this time. YT please D:
Anyway, thank you so so much for your help! You are such a lifesaver.
I am aware of IDEAS. However, the technical officer who gave me some training to operate ImageStream told me the software is difficult to use (She doesn't even know how to use it herself). So I opted for alternatives. Of which I stumbled across your channel, and FlowJo seemed simple and intuitive enough so I decided on FlowJo. After reading your advise I'll definitely give IDEAS another go. Hilariously enough, even though my uni has a pretty advanced flow cytometer (The ImageStream), very few people, including the technical officer herself, know how to operate the instrument to its full potential.
The only unfortunate thing is that I only acquired my data in .fcs format, and not in the .rif format that IDEAS mainly recognize. I tried to import my .fcs data into IDEAS but nothing really happened (Sometimes the software even crashes lmao). I'll have to run my experiments again but its not a big deal because I just did a trial run to get familiar with the process. In the meantime I'll mess around with FlowJo first.
Again thank you so much for your help! I really appreciate your time and effort. Hope you have a nice weekend!
Hi Aja, I am having difficulty compensating my annexin-FITC stained cells.FlowJo is new for me. I have used FACSDiva a few times, Your samples look great. Mine seem uncompensated, and I watched so many UA-cam videos for compensation, however, it does not work. I am not able to see the Annexinv-FITC-A parameter, I have FITC only, also No PI, only PE.
I have done Annexin-V apoptosis with 16 different samples:
- unstained (no drug treatment), annexin v-FITC only (no drug treatment) , PI only ( no drug treament ), PI and annexin-vFITC (no drug teatment ).
-unstained (250 drug treatment), annexin v-FITC only (250 drug treatment), PI only (250 drug treatment), annexin v-FITC and PI (250 drug treatment)
-unstained (125 drug treatment), annexin v-FITC only (125 drug treatment), PI only (125 drug treatment), annexin v-FITC and PI (125 drug treatment)
-unstained (H202 treatment), annexin v-FITC only (H202 treatment), PI only (H202 treatment), annexin v-FITC and PI (H202 treatment)
ONLY the first 4 are for compensation ?? I am not sure how to analyze the data. Could you please lease please help ? Can I please send the FCS files to your email please?
I got your email- just replied back!
@@ajarieger_flow THANKS VERY MUCH. You are an amazing scientist and even more amazing and incredible as a person.
Thank you so much for all the informative videos on FlowJO.
I have a question I am trying to check the mitochondrial membrane potential of cells for which I am using JC-1 stain. It will tell about two populations.
How to do an analysis for that?
Which filter to use?
Do I need to do the analysis in FL1-FITC and FL2 PE-A?
I am confused.
Kindly help.
Happy to help! JC-1 will be "red" when the mitochondria are active and "green" as they lose membrane potential. Both will be excited off at 488nm laser on most flow cytometers. The RED fluorescences at 590nm; the GREEN at 529nm. Not knowing the setup of your instrument, I don't know channels they will be in. But look at the setup and see which channel will capture this emission. Hope that helps!
@@ajarieger_flow Thank you so much.
I have already acquired the data. I want to know how to analyze the data and differentiate bw two different populations with HMMP and LMMP.
Thank you so much in advance.
I use ImageJ for the analysis.
exciting 😍
Hi Aja, I am doing my first-time flow cytometry analysis using flowjo. But I am not able to see the BL1-A ::Annexinv-FITC-A option in my screen. instead of that I AM seeing CoMP-FL1-A :: FL1 INT LOG , this option. Also i have a doubt, I have done flow cytometry with 8 samples..... unstained( no drug treatment ), annexin v only ( no drug treatment ) , PI only ( no drug treament ), PI and annexin v (no drug teatment ). unstained ( with drug treatment), annexin v only ( with drug treatment ), PI only ( with drug treatment), annexin v and PI ( with drug treatment) So here first 4 are for compensation ?? I am not sure how to analyze the data. could you please help to give some idea.
Hi! Without seeing the data, it is hard to say for sure. However, this indicates to me that you compensated when you acquired the samples and that’s why they show up as “COMP…”.
For your compensation, I would recommend using the treated samples so you have a mix of alive and dead cells- you will need both for compensation.
Thanks for the quick response. As this is my first time with flow cytometry I am having a lot of trouble with the analysis. IF possible can I share you my data?
@@Livelife4701 yes you can email me at aja@ualberta.ca
Thanks for your explanation!