Thank you for the informative video :) I have been doing bacterial cell sorting and it has been much much harder to find resources for bacteria vs mammalian cells. It would be really useful if you made a video on this or add links to resources for bacteria
Dr Aja I was in a flow cytometry workshop and Joanne lanigaan was the speaker she mentioned you in her presentations and I was so proud of you and almost shouted that's my beautiful online tutor ❤
dear Dr Aja, this is lin from Shanghai, China, a big fan of your channel，which helps me lot. I am a doctoral candidate majoring in life science and technology and doing research in the field of hematopoietic stem cells (HSC). I have a problem that has haunted me for a while but haven't found a resolution. I have a chimeric mouse model in that cells could show GFP and tdtomato, respectively. I have to compare several phenotypes, like ROS, mitochondria membrane potential, protein synthesis rate, and other protein expression levels, in these two cells, and all these indexes should be detected in the APC channel. The staining panel for HSC is lineage-AF700, Scal1-PEcy7, cKit-APCcy7, CD150-BV711, CD48-BUV395, CD34-BV421, GFP, and tdtomato. The problem is when I compare the APC signal in two different mice, it seems that the compensation between AF700, APCcy7, and PEcy7 signals was also changed with the changes of APC signal intensity. It seems unreasonable to compare the signal of one channel in different samples with different compensation. Then the results could be altered in terms of the compensation used. So could you tell me how can I resolve this issue? Or if there is any flow cytometry basic acknowledgment I should know to help me understand this problem and solve it. Thank you very much for your patience and hope I can get your response.
Thanks- I'm glad it's been helpful!! A few things to make sure you are considering: 1. even though all your dyes are in the APC channel, make sure you are compensating them each individually with the dye that is used in the experiment (i.e. don't use ROS staining to compensate for membrane potential). Each of these dyes will have a unique spectrum and will need to be treated as such. 2. it is normal for compensation values to fluctuate over time. They will never be identical, even on samples run with the same fluorochromes, on the same instrument, with the same setup. Knowing this, and assuming you are correctly matching dyes during compensation, there is no issue with comparing values between experiments that have different compensation values. The compensation is merely correcting for the channel overlap that occurred that day. You will be worse off trying to use the same compensation for all samples and comparing those- this is much more likely to introduce artifacts and inaccurate results into your data. Hope that helps!
Dear Aja, Currently I have been stuck with FACs sorting because I am getting fewer cells. I am trying to sort endothelial cells from a mouse heart. I use the FAC sorting core facility of my university to do this. I take my antibody-stained samples to them and they sort for me. I have a doubt that they are using the wrong gating strategy for DAPI and as a result, I am getting fewer sorted cells. If you are okay, I want to show the DAPI gating strategy to you to make sure. If you are okay, I would like to show you the gating strategy of DAPI. I can send it to you via email. Thanks for answering my questions.
![Adjusting compensation: how to handle a "bad" matrix [FlowJo advanced]](/img/n.gif)
Amazing !! Thanks so much for share this knowledge
Thank you for the informative video :) I have been doing bacterial cell sorting and it has been much much harder to find resources for bacteria vs mammalian cells. It would be really useful if you made a video on this or add links to resources for bacteria
Dr Aja I was in a flow cytometry workshop and Joanne lanigaan was the speaker she mentioned you in her presentations and I was so proud of you and almost shouted that's my beautiful online tutor ❤
This made my day! Thank you and glad the videos have been helping 😊
Any topics you want covered just let me know!
Amazing!
I have no idea what is going on here, but I watch every video.
It's a flow cytometry party!
dear Dr Aja, this is lin from Shanghai, China, a big fan of your channel，which helps me lot. I am a doctoral candidate majoring in life science and technology and doing research in the field of hematopoietic stem cells (HSC). I have a problem that has haunted me for a while but haven't found a resolution. I have a chimeric mouse model in that cells could show GFP and tdtomato, respectively. I have to compare several phenotypes, like ROS, mitochondria membrane potential, protein synthesis rate, and other protein expression levels, in these two cells, and all these indexes should be detected in the APC channel. The staining panel for HSC is lineage-AF700, Scal1-PEcy7, cKit-APCcy7, CD150-BV711, CD48-BUV395, CD34-BV421, GFP, and tdtomato. The problem is when I compare the APC signal in two different mice, it seems that the compensation between AF700, APCcy7, and PEcy7 signals was also changed with the changes of APC signal intensity. It seems unreasonable to compare the signal of one channel in different samples with different compensation. Then the results could be altered in terms of the compensation used. So could you tell me how can I resolve this issue? Or if there is any flow cytometry basic acknowledgment I should know to help me understand this problem and solve it. Thank you very much for your patience and hope I can get your response.
Thanks- I'm glad it's been helpful!! A few things to make sure you are considering:
1. even though all your dyes are in the APC channel, make sure you are compensating them each individually with the dye that is used in the experiment (i.e. don't use ROS staining to compensate for membrane potential). Each of these dyes will have a unique spectrum and will need to be treated as such.
2. it is normal for compensation values to fluctuate over time. They will never be identical, even on samples run with the same fluorochromes, on the same instrument, with the same setup.
Knowing this, and assuming you are correctly matching dyes during compensation, there is no issue with comparing values between experiments that have different compensation values. The compensation is merely correcting for the channel overlap that occurred that day. You will be worse off trying to use the same compensation for all samples and comparing those- this is much more likely to introduce artifacts and inaccurate results into your data.
Hope that helps!
@@ajarieger_flow It certainly helps a lot, thank you for your reply and hope to watch more your tutorial videos. Having a nice day❤
Dear Aja, Currently I have been stuck with FACs sorting because I am getting fewer cells. I am trying to sort endothelial cells from a mouse heart. I use the FAC sorting core facility of my university to do this. I take my antibody-stained samples to them and they sort for me. I have a doubt that they are using the wrong gating strategy for DAPI and as a result, I am getting fewer sorted cells. If you are okay, I want to show the DAPI gating strategy to you to make sure. If you are okay, I would like to show you the gating strategy of DAPI. I can send it to you via email. Thanks for answering my questions.
Yup feel free to send my way. aja@ualberta.ca
@@ajarieger_flow I just sent it. Thanks
I can't get my correct apoptosis data😭☹
What’s happening? What issues are you having?
@@ajarieger_flow I think there may be a problem with the experimental operation, not the data processing😩，the positive groups didn't show apoptosis🤕
@@xxy-ix5qm what are you using to stain your cells? And what’s your positive control?
@@ajarieger_flow we bought Annexin V-FITC assay kit （Annexin V-FITC/PI) and the apoptosis positive reagent
@@xxy-ix5qm and you’re using the AnnexinV binding buffer? Diluted with water?