So basically, I am depleting lineage positive population from isolated bone marrow cells using MACS and then staining the lineage negative population with cocktail of fluorochrome tagged antibodies for Streptavidin, cKit, Sca1, CD150, CD48, CD34, CD16/32, IL7Ralpha, Live/Dead Stain (Ghost Viability Dye, TONBO). I did my earlier experiments using two combination of cocktails using (APC, APC-Cy7, PerCpCy5.5, FITC, PE, BV711, Pacific Orange), but for my future experiments I want to do it as a 9 color experiment. As I am looking for a very rare population, I want to find out what multicolors are best to use in combination and would aid in giving better analysis of rare events.
I used LSR Fortessa X-20 analyzer for my earlier experiments, single stained controls were good only a slight problem in Streptavidin-APC
Hi Bhakti, you are on the right track with spreading your colors across multiple lasers. For your 9 color experiment, you can do 1-2 UV dyes, 2-3 violets, 2 colors excited by 488 (usually these are FITC and PerCP-Cy55 or analogs), 2 YGs and 2 reds.
I would need way more details from you go give you specific answer... also, your flow core is amazing, you should get in touch with them to hear their advice on panel design.
Anna C Belkina is correct, utilize your flow core.
Also, I use the following to make my panels and then check them with my core.
https://www.biolegend.com/instrument_guide
https://www.biolegend.com/panelselector
Hi, I think the above answers are absolutely correct: it depends on the setup of your machine and you can use the Biolegend tool. However, I think that 9 colours with FACS would be hard to analyse anyway; have you already thought about CyTOF, maybe in a collaboration setting?
Andrea Tavosanis, it's not 2010 anymore :) 9 colors is trivial with flow cytometry. With their instrument (X20), I say anything under 15 colors should be pretty easy. Also CYTOF+very rare subset = lots of pain...
Anna C Belkina that's great to hear. I will set up similar bone marrow analysis experiments with less colors but also with a less advanced machine. I figured that dealing with compensation would be challenging with so many colors, but if that's not the case there should be no issues. I also read and thought that CyTOF doesn't really care about rare populations, as long as the individual signal (eg antigen expression) is strong enough; have your experiences been different?
CYTOF is way slower and recording a decent size population is hard (and expensive). People often record
Thanks everyone for your answers.
Anna: Thanks again Anna. I am looking for LSK, HSC, CMP, GMP, MEP, CLP population in mouse bone marrow cells to be specific and HSCs are very rare, usually (3-10%). So I am planing to use APC, APC-Cy7, FITC PE, Pacific Blue, Pacific Orange, PerCP-Cy5.5, BV711 and PE-Cy7 next time.
1. Are my colours OK?
2. I ave used Biotin Rat anti mouse antibodies to deplete lineage positive population using MACS before FACS progenitor staining. And for confirming lineage population depletion, I am including streptavidin to differentiate between lineage positive and negative population. However, last three times I had faced problems with single stained controls (I am using Invitrogen Ultracomp Compensation beads for single stained controls) I don't understand why I only see one peak and I am not able to see two distinct peaks in single stained control for Streptavidin colour (Streptavidin-APC). I even tried using other streptavidin colour (Streptavidin-PECy7) but faced similar problem, got only one peak.
3. Could you/anyone suggest me better way to gate my lineage negative population ?
Hi Bhakti Patel - if you're still looking for help I helped create a full protocol for mouse bone marrow phenotyping. Specifically for your point #3 this protocol shows gating suggestions for gating out lineage positive cells (in this case using the FITC channel) and suggestions for panels to identify LSK, LSK/SLAM and ESLAM phenotypes.
Take a look and let me know if you have any questions if you're still having trouble. https://www.stemcell.com/flow-cytometry-methods-for-identifying-mouse-hematopoietic-stem-and-progenitor-cells.html?utm_source=researchgate
Cheers!
Amanda
You probably have this sorted out by now, feel free to get in touch in future if you'll have difficulties in designing a larger panel, we might help out.
Thanks Amanda and Kristian.
Yes I have optimized the 9 colour panel. Thanks everyone for your help.
Now I am trying to optimize 11-colour panel. Any suggestions for 11 colours that could be used together (My 9 colour panel includes above APCCy7, APC, FITC, PE, PerCpCy5.5, PE-Cy7, BV711, Pacific Orange and Pacific blue)?
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