Thank you so much Nathan! We are happy you're enjoying it!

You are very welcome!

If you are using a BD machine and use the CS&T settings this is a good starting point especially if you are using lymphocytes but not everybody does! Sometimes the settings that CS&T gives are not appropriate for other cell types and that's where the voltration can be useful. I also find that with some cells, particularly when using far-red emitting fluorochromes that we can sometimes get a better separation by changing the voltages from those suggested by CS&T. You are right that we should make sure we are in the linear range of each detector and this is also a figure given by the CS&T program. In practice, I try not to have any signal above 10^5 when using Diva.

Hi Carlos! Great question. This is something that may be easy to identify as debris or background as a cytometrist but not so easy if you’re not used to looking at this data all the time. We know in this dataset there is one homogenous population of cells by scatter we expect. This population is clearly identified and pulled out from the population that is very low on FSC and SSC which we typically identify as debris. What can be really beneficial if you’re unsure is to start off by looking at your fluorescent population of interest and gating on the positive cells (if there are any) and from there looking at a FSC/SSC plot to see where these events fall to help you set a gate for scatter. Another option would be to include a live cell marker such as calcein. Some messy tissue preps have lots of debris (think brain!!) and we can pull out metabolically active live cells from debris by using calcein, as it will be cleaved by esterases and fluoresce in live cells but not debris. Another option for you would be to run a sample that was buffer alone. You will see a lot of this debris and background there. Since you know there are no cells in this sample, you would expect all cells to be higher in scatter and can use this buffer alone control as a guide until you become used to scatter gating. I hope this all helps! - Kathy

@@kathydaniels1077 Thank you very much for your answer!! Everything's clear now! Amazing tutorial!

Hi Asisipo. We covered voltage optimization in another OpenFlow class: kzfaq.info/get/bejne/jM6ljaeBu56ZeIk.html. Check that video out and let us know if you have any questions. :)

@@RuiGardner Good day Dr. Gardner Thank you for your response and the video recommendation. The video has been very helpful when trying to set up the voltages for my panel, however, I got a bit confused, my panel has 12 colours, so I was wondering if I need one tube (cells and one fluorochrome) for each colour or if I can use one tube with all the colours mixed to set up the voltages? Another confusion was whether I need a specific number of cells (PBMCs) for each voltage run or if I just need enough for about 10 000 events? Thank you.

Hi Jennifer, Thanks for the question.The questions I would have for you is which cytometer have you used to acquire data and which version of the Flow Cytometry Standard is your data? Most cytometers will produce data files in the FCS3.0 format where time is added as a keyword and then should be available to you in the analysis software as the final parameter in the list.

​@@DerekDaviesCytometry Thanks so much for your reply. My data is from MACSQuant Analyzer 16 and I'm using FlowJo V 10.8.1. The FCS files are version 3 and when I inspect a sample it does show the boxes for median parameters vs. time but there is not time option to actually open it up on x or y axis to cut out points where I know the machine clogged.

@@JenniferGrinder Hi Jenner! Would you mind sending an example fcs file to openflowcytometry@gmail.com? We can take a look for you and see if we can find out a solution. Thanks! - Kathy

@@OpenFlowCytometry I appreciate all your help! I figured it out, "time" in MACSQuant language is HDR-T, so we are all good. :)

@@JenniferGrinder Wonderful, happy to hear! A good start to 2023!

Flow cytometry allows us to correlate the presence/absence and level of signal. Although we dont get an image and cant correlate fluorescence signatures within cells, we can certainly say which cells are expressing which marker. So in the case of FITC/PE we would potentially be able to see 4 populations - a double negative (no expression of either marker), a double positive (cells expressing both markers) and two single positives (cells that either have PE OR FITC), we can do this because the cytometer is making single cell measurements.

​@@DerekDaviesCytometry My concern is that the SEQUENTIAL application of multiple lasers to homogeneous cells cannot distinguish the protein location within a cell, unlike imaging using a fluorescence microscope. The FACS result can only say whether this cell expresses protein or not. This aspect makes FACS is not a perfect tool for qualitative analysis. Imagine that an "A" protein-FITC can exist in the two different cellular compartments such as ER and Golgi. If you want to sort these heterogeneous cell populations into two groups (ER-localized "A" and Golgi-localized "A"), you may want to co-label cells with either ER or Golgi marker-PE. If you use a fluorescence microscope, you can easily distinguish which cells express "A" in the ER or Golgi. However, with the same sample, FACS results just tell you those cells are all positive for FITC and PE because ER and Golgi are always within the cell. So I am just curious if two or more different excitation lasers and detectors are technically available, we could resolve the aforementioned problem.

@@rangeo8783 Thanks for the comment! You are correct in that you (usually) cannot get localization information with flow cytometry. In conventional flow cytometry, if you have cells expressing GFP, you cannot tell where the fluorescent protein is in the cells, just that it is present. There are certain fluorochromes (like pHrodo) that are nonfluorsecent if they are on surface of cells, but become fluorescent upon exposure to low pH environments. This enables you to determine if internalization has occurred. I said usually in my statement earlier because there are imaging flow cytometers that are on the market that allow for you to get morphological information about your cells (via brightfield) in addition to fluorescence output. I'd suggest you do a search on "imaging flow cytometers" and check it out. The Amnis is one example that takes many digital images of the cells. Think of it as a hybrid microscope and flow cytometer in a way. If conventional flow cytometers are your only available option and the same exact protein is expressed on both ER and Golgi for example, you would not be able to differentiate expression. I would suggest seeing if you can identify monoclonal antibodies that are specific to either the ER or the Golgi complex. Then you can have one Ab conjugated to one fluorochrome and the other Ab conjugated to another (Er-FITC / Golgi -PE) so you can determine if they are present without the localization information. Flow cytometry is an incredibly powerful tool, but it is not always the method you will want to proceed with. You have to ask yourself what is your biological question and then from there determine the best approach to answer that question. Hope this helps! Kathy

@@OpenFlowCytometry Thank you for your kind and detailed answer. The conclusion is we cannot distinguish the subcellular location of protein even if I use multiple fluorescences with conventional flow cytometry! Amnis sounds excellent. However, I am still curious if it could be applicable for high-throughput screening, like an advantage of primay flow cytometry function. I appreciate your great help and suggestions.