Question on the gating of CD8 cells at around 1:10:07. I saw there is a diffusion of positive CD8 cells and negative cells. How did you know the position of gating is correct? Or not moving down a little bit to gate the correct plot? Thanks.
Hi Fei Tu, thanks for a great question! For this CD8, you do see a moderate and a high expressing population. For this experiment, there was no biological question I was trying to answer, but merely use these samples as an educational tool for full spectrum flow cytometry. I gated on the high expressing cells arbitrarily. If I was trying to gate on any cells that were CD8 positive, regardless of intensity, I would have used an unstained sample or a CD8 BV421 FMO sample if I saw any issues with spreading. Sometimes scientists are looking to pull out different levels of expression and may gate there samples a little differently. There are certain markers that are expressed at a continuum or have different intensities. What you look and how you gate these is going to require you to think about your biological questions. Hope this helps! I'm sure Derek or Rui will chime in if they have any additional comments. :-) - Kathy
@@feitu6403 It is a great question as deciding what is positive for a particular marker is crucial to be able to answer your biological question. As Kathy says, in most multicolour experiments we would use an FMO (Fluorescence Minus One) to decide what is positive - however there is still a degree of subjectivity as to where to set that marker - do you set is such that absolutely 0% of events are +ve in the FMO, or are you happy to set it to be 0.1%/0.5%/1%? I would always look at the staining pattern and think about the biology.
excellent video about full spectra flow cytometry. Here is a little question I have for a single stain for compensation. From your other video for classical flow cytometry, I learned that the beads and cells are used for each staining. In Aurora, there is a method to remove the autofluorescence of beads or cells. However, in the classic flow, I used beads for a color, and cells for another color, how the machine or Diva (BD company provided) we used to remove autofluorescence of beads and cells for the samples. Looks forward to your comments. Thanks.
Hi Fei. Another great question! When unmixing in SpectroFlo, you have an option when setting up the experiment to choose whether the controls are beads and cells so that the software uses the correct autofluorescence to unmix. You also have the option to run an unstained cell and an unstained bead sample. This does not remove autofluorescence of beads or cells, but uses it to correctly unmix. If your experimental samples have high autofluorescence which impacts your experiment, there is an option when unmixing "Autofluorescence as a Fluorsecent tag" (watch here for detailed instructions) ua-cam.com/video/Gg-m3OAY9-Y/v-deo.html. This reduces the impact of autofluorescence for your experiments. Thanks! - Kathy
Great webinar, thank you! Is it possible to use a separate unstained control for spectral unmixing and autofluorescence extraction? For my current panel, we're using murine lung cells as reference controls (going to try UltraComp beads but haven't yet). Full stained samples are murine adipose, DRG, TG, and WBCs. We do not have enough cells from each of these tissues to run reference controls using them. Any advice is much appreciated!
You are very welcome! ua-cam.com/video/4uaMCv3tzUs/v-deo.html This video by Cytek shows at about 1:30 how you can create reference controls, specifically showing that you will have the unstained control which is the cell type you will have as your experimental samples. The reference controls themselves can be beads or cells, and you can either draw the negative gate for each of these controls or specify a negative control for them. You have the option to define additional negative controls as well, so if you have a different cell or particle type you are using for reference, you can add a negative control of that same sample type here. Another important video is here: ua-cam.com/video/51Ek2KgtxY8/v-deo.html If you are running multiple types of experimental samples (different tissues with different autofluorescence) you will need to account for that. The video referenced shows how you can run multiple tissue types in the same experiment, accounting for the heterogeneity in autofluorescence. Hope this helps and happy flowing! - KD
@@OpenFlowCytometry Thank you so much! Also, I found the MSKCC protocol for Aurora start up and shut down. I noticed during the 40 minute warm-up period before QC that you perform 30 minutes of initial cleaning on high. Our facility does 30 minutes of running DI water on high (our facility just received the Aurora so we're still trying to mold our SOPs). Is there reason to switch to cleaning for 30 minutes? If so, what concentrations of bleach and detergent do you use for the clean and rinse steps? Much appreciated!
@@kaylaparr7732 Hi Kayla! You are very welcome. Typically we run the cleaning as a precaution for all of our instruments at the beginning of the day. If water works well for your facility, that's great! We have run 10% bleach, FACSRinse and then water. Rinse is no longer being sold, so another potential alternative is Coulter Clenz. Thanks! - KD
@@OpenFlowCytometry Thanks again! I think we may try it as the supervisor noted some problems he thinks are being caused by bad samples going up the fluidics. There are just so many resources to comb through to see what works for us.
Question on the gating of CD8 cells at around 1:10:07. I saw there is a diffusion of positive CD8 cells and negative cells. How did you know the position of gating is correct? Or not moving down a little bit to gate the correct plot?
Thanks.
Hi Fei Tu, thanks for a great question! For this CD8, you do see a moderate and a high expressing population. For this experiment, there was no biological question I was trying to answer, but merely use these samples as an educational tool for full spectrum flow cytometry. I gated on the high expressing cells arbitrarily. If I was trying to gate on any cells that were CD8 positive, regardless of intensity, I would have used an unstained sample or a CD8 BV421 FMO sample if I saw any issues with spreading. Sometimes scientists are looking to pull out different levels of expression and may gate there samples a little differently. There are certain markers that are expressed at a continuum or have different intensities. What you look and how you gate these is going to require you to think about your biological questions. Hope this helps! I'm sure Derek or Rui will chime in if they have any additional comments. :-) - Kathy
@@kathydaniels1077 Appreciate your patient and detailed explanation. Fei
@@feitu6403 It is a great question as deciding what is positive for a particular marker is crucial to be able to answer your biological question. As Kathy says, in most multicolour experiments we would use an FMO (Fluorescence Minus One) to decide what is positive - however there is still a degree of subjectivity as to where to set that marker - do you set is such that absolutely 0% of events are +ve in the FMO, or are you happy to set it to be 0.1%/0.5%/1%? I would always look at the staining pattern and think about the biology.
excellent video about full spectra flow cytometry. Here is a little question I have for a single stain for compensation. From your other video for classical flow cytometry, I learned that the beads and cells are used for each staining. In Aurora, there is a method to remove the autofluorescence of beads or cells. However, in the classic flow, I used beads for a color, and cells for another color, how the machine or Diva (BD company provided) we used to remove autofluorescence of beads and cells for the samples. Looks forward to your comments. Thanks.
Hi Fei. Another great question! When unmixing in SpectroFlo, you have an option when setting up the experiment to choose whether the controls are beads and cells so that the software uses the correct autofluorescence to unmix. You also have the option to run an unstained cell and an unstained bead sample. This does not remove autofluorescence of beads or cells, but uses it to correctly unmix. If your experimental samples have high autofluorescence which impacts your experiment, there is an option when unmixing "Autofluorescence as a Fluorsecent tag" (watch here for detailed instructions) ua-cam.com/video/Gg-m3OAY9-Y/v-deo.html. This reduces the impact of autofluorescence for your experiments. Thanks! - Kathy
Great webinar, thank you! Is it possible to use a separate unstained control for spectral unmixing and autofluorescence extraction? For my current panel, we're using murine lung cells as reference controls (going to try UltraComp beads but haven't yet). Full stained samples are murine adipose, DRG, TG, and WBCs. We do not have enough cells from each of these tissues to run reference controls using them. Any advice is much appreciated!
You are very welcome! ua-cam.com/video/4uaMCv3tzUs/v-deo.html This video by Cytek shows at about 1:30 how you can create reference controls, specifically showing that you will have the unstained control which is the cell type you will have as your experimental samples. The reference controls themselves can be beads or cells, and you can either draw the negative gate for each of these controls or specify a negative control for them. You have the option to define additional negative controls as well, so if you have a different cell or particle type you are using for reference, you can add a negative control of that same sample type here. Another important video is here: ua-cam.com/video/51Ek2KgtxY8/v-deo.html If you are running multiple types of experimental samples (different tissues with different autofluorescence) you will need to account for that. The video referenced shows how you can run multiple tissue types in the same experiment, accounting for the heterogeneity in autofluorescence. Hope this helps and happy flowing! - KD
@@OpenFlowCytometry Thank you so much! Also, I found the MSKCC protocol for Aurora start up and shut down. I noticed during the 40 minute warm-up period before QC that you perform 30 minutes of initial cleaning on high. Our facility does 30 minutes of running DI water on high (our facility just received the Aurora so we're still trying to mold our SOPs). Is there reason to switch to cleaning for 30 minutes? If so, what concentrations of bleach and detergent do you use for the clean and rinse steps? Much appreciated!
@@kaylaparr7732 Hi Kayla! You are very welcome. Typically we run the cleaning as a precaution for all of our instruments at the beginning of the day. If water works well for your facility, that's great! We have run 10% bleach, FACSRinse and then water. Rinse is no longer being sold, so another potential alternative is Coulter Clenz. Thanks! - KD
@@OpenFlowCytometry Thanks again! I think we may try it as the supervisor noted some problems he thinks are being caused by bad samples going up the fluidics. There are just so many resources to comb through to see what works for us.
very nice, thank you