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Facility News
Summer ☀️ is coming to an end and we are all slowly coming back to our routine. I hope that you managed to disconnect for a few weeks and gather all the sunlight you could !
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We are starting the new "school year" with a day long workshop on October 15th about imaging flow cytometry and cell sorting (DETAILS HERE). As some of you may know, we acquired a BD Discover S8 spectral sorter recently and it offers new options for your experiments. Please join us for a morning presentation followed by an hands-on session in the afternoon ! Contact us if you'd like to submit some samples !
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In this month FACS Tips, Kevin is answering some of the questions we keep asking ourselves as we are troubleshooting flow cytometry experiments days after days. Take a look !
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Leila Hadadi won the mug this month, Congratulations to you !
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Each month, we will ask you 3 questions about the newsletter topic. If you win, you can enter the lottery to win a unique mug designed by the FCF team !
Please take few minutes to answer the quiz HERE.
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FACS Tips
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Oh, No! I added the wrong antibody! And Other Troubleshooting Scenarios
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As I stock up on new topics for the start of the upcoming school year, this month I’m going to highlight some fun hypothetical situations and their outcomes. These are all taken from a fantastic flow cytometry textbook, Flow Cytometry Basics for the Non-Expert, written by Christine Goetz, Christopher Hammerbeck, and Jody Bonnevier. I think this is a great resource for many of your flow cytometry questions. It’s written simply and thoughtfully to support both your theoretical understanding, but also practical guidance, helping you navigate from experiment design to data analysis.
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Below I’ll break down three of their highlighted situations, but if you're curious for more, I highly recommend checking out the textbook. I have a PDF copy that I’d be happy to share—just let me know if you're interested!
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I Added the Wrong Antibody—Can I Wash It Out Quickly?
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This is a pretty easy to imagine situation. You’re staining multiple panels, each with 12 to 15 colors, and you accidentally add the wrong antibody to your sample. Can a quick wash save the day?
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The authors tested this question by tracking the speed of antibody binding to human PBMCs. By evaluating a CD4 APC stain after time points from 10 seconds to 30 minutes, they could clearly see that even after only 10 seconds of incubation, there was significant positive staining for APC. While the full 30 minute incubation produced 61.3% of CD4+ T cells from lymphocytes, even just 1 minute of staining produced 58.2% of the same population. I certainly wouldn't suggest shortening your staining incubation time to 1 minute, 30 minutes produced the highest MFI with best separation, but this demonstrates that a quick wash won’t undo the mistake. You'll need to start over.
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* The one caveat to this is that if it's an isotype control you accidentally add, then yes, you can continue with staining in the same tube.
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Wouldn’t it be quicker if I added my primary and secondary antibody at the same time?
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We all want to save time in our experiments, so sure, let’s do 1 wash when it says 3, or skip that blocking step, what damage can it do? I guess this is a slightly more extreme corner cutting measure than those, but what if you add your primary and secondary antibody simultaneously. Will it still work?
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Sort of, but not really*.
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Adding both primary and secondary at once risks the two forming a complex before the primary binds to its target, which can compete with the binding process. In the authors’ test, this resulted in a one-log drop in staining intensity compared to the standard sequential protocol.
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*The authors performed a similar test, but with Biotin and Streptavidin antibody pairs. Interestingly, when the streptavidin secondary is added subsequently or simultaneously to the biotin primary, the detection of the population of interest doesn’t change. This was true for both the percentage and MFI of the population. The explanation for this is, streptavidin only has specificity for the Biotin, while another secondary antibody that could be goat anti-mouse, goat anti-rat, etc., will bind any antibody from a certain species. There is still the issue of antibody complex formation interrupting the ability of the primary to bind, so while in this example it didn’t have any effect, that may not be true of all situations.
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I Added the Perm Before the Fix—Is My Sample Ruined?
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Again, this isn’t the hardest situation to imagine, we all make mistakes on a busy day. If we find ourselves in this spot though, can anything be salvaged?
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Unfortunately, you’ve killed a lot of your cells at this point. In a test by the authors, they added a perm buffer to a sample of PBMCs then washed them immediately to try and minimize the damage. When compared against properly fixed and permed PBMC samples, using both naive and stimulated, the FSC and SSC plots showed that the cells shifted into the debris/dead cell region. Once this happens, there’s no recovering the sample.
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I’m sure these answers aren’t all that surprising, we follow protocols for a reason and it’s to avoid the mistakes that come about when things fall out of order, or mistakes are made. But it is fun to see these mistakes played out and broken down, and understanding why these mistakes are problematic can deepen our appreciation for protocols.
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Again, I highly recommend this textbook for anyone looking to bolster their flow cytometry skills. Also, if you have any hypothetical (or real) questions along these lines, send them to the FCF staff and we can see if we can do some tests of our own to report back.
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