8
votes
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Magnetic-activated cell sorting vs. FACS
Magnetic-activated cell sorting (PDF download), or MACS, is a procedure developed by Miltenyi Biotec to separate cells from complex mixtures using antibody-coated magnetic nanoparticles. The ...
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3
votes
Flow Cytometry Channels
I'll amend this answer as I get details from you.
What is your cytometer, and how are the optics set up? You typically have 1-5 lasers: UV (355nm), violet (405nm), blue (488nm), green/yellow (~561nm) ...
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3
votes
Accepted
Staining cells for FACS at 4 degrees or ambient temperature
You have several questions, please try to limit your posts to one question each. That being said, I'll try to address each of your concerns.
Does the cold cause these cells to apoptose?
No. The ...
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2
votes
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Cell dye in red fluorescence spectrum
I've used the ThermoFisher brand of CellTrace dyes. Specifically, I co-cultured two populations of cells, one with CellTrace Far Red in the APC channel and another with CellTrace Yellow in the PE ...
- 8,081
2
votes
How are single cells sorted in an scRNA-Seq protocol by FACS?
we don't know the baseline of CD markers' expression levels yet?
There are standard settings that can be used between runs that will give consistent results from one sample to the next. There are ...
- 14.1k
2
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How are single cells sorted in an scRNA-Seq protocol by FACS?
I will append the previous sentence in your protocol, and put what might help you in bold:
To obtain B cells, NK cells and monocytes, a splenocyte suspension was stained with PE-Cy7-...
- 6,124
1
vote
Number of membrane proteins of a particular type per cell
If you know the stoichiometry of a particular ion channel and do something like patch-clamp, where you know the surface area being clamped, then you can measure the ion-flux per unit time and from ...
- 9,024
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Can I use fluorophore conjugated antibody as neutralizing antibody?
In short, yes, most likely. When an antibody binds to the antigen at a region (epitope) that is needed for the antigen's activity then the activity is reduced ("neutralized"). The (proper) ...
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1
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CRISPR-Cas9 knockout
To answer your question multiple parameters which one should be taken into account.
There are two main sets of parameters: gene specific and cell line specific.
Parameters related to your specific ...
- 620
1
vote
Accepted
Non-nucleated cell-like population with RNA
Maybe you are seeing extracellular vesicles1?
At least might be worth checking out since extracellular vesicles containing RNA have been reported within the hemolymph of mosquitos2.
References:
1: ...
- 5,513
1
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Flow cytometric counting of apoptotic adhering cells
Here some resources,
Cell harvesting effects on Annexin V staining.
Protocol for apoptotic analysis using Annexin V.
Quite a long discussion about this issue.
Another protocol.
Cheers,
Pedro
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Is it possible to sort droplets inside an oil phase with a FACS
It's possible that there's some specialized modified machine that can do it, but conventional flow cytometry machines can't do it. This article suggests one possible approach:
Unfortunately, FACS ...
- 14.1k
1
vote
Accepted
Flow Cytometry to learn about Cell cycle
The basis of analysing flow cytometry data in regard to the cell cycle is the fact that DNA synthesis occurs in S-phase and therefore cells before S-Phase (G0/G1) have half the amount of DNA compared ...
- 4,356
1
vote
Accepted
Flow cytometry data Analysis
Disclaimer: Assumes you know how flow cytometry works.
You've done a flow-based AnnexinV apoptosis assay here.
Annexin A5 (AnnexinV) protein binds to phosphatidylserine (PS), which is typically in ...
- 8,081
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Cell dye in red fluorescence spectrum
There is a number of different dyes possible, as you can see in this figure (from here):
A good overview can be found in the "Selecting Reagents for
Multicolor Flow Cytometry" application note ...
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1
vote
Accepted
Purpose of antibody wash
Two reasons for antibody wash:
To limit non-specific epitope binding. All ABs have specificity. Washing is usually trying to remove weaker binding to non-intended targets.
For flow, excess AB that ...
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