It really depends on your application. Are these THP1s differentiated? If so, the process of removing the adherent cells may change their light scatter properties, making me lean towards the more more liberal gate. However, generally speaking I go with tighter gates. Have you looked to see if those cells excluded in the tighter gate have the same marker or cytokine expression?

It looks like you may have a second population of cells around 180k(Fsc). Using a contour plot might help determine this.

I would go with tighter gates (even tighter than your 67% gate). The events with higher Fsc/Ssc makes me think they are doublets; are you removing them?

Hi Christopher, yes, they are THP-1 cells. For detachment, I use TrypLE (Gibco). What do you use?

Hi Evans, I checked and those are not doublet cells.

Regarding the method for removing the adherent cells, there was a discussion on the cytometry mailing list last year about different methods and the potential for trypsin to hydrolyze surface proteins. Some people suggested chelators, like EGTA along with gentle manipulation with a rubber spatula. Someone also suggested fixing before trypsinization.

Here's a link to the thread:

http://goo.gl/iVvrn

click on messages sorted by thread to read all of the replies.

Hi Abdullah!

I usually go with wider gates, but by wide I mean as your 67% gate. For me, a tighter gate, as Evan suggests, should include just the population near the green dots border. It depends on what your are looking for. If you know your population is very homogeneous, you shoud use tight gates; but if you are expecting some variability you may loose important events (especially if you are analysing rare events) with tighter gates.

About the detachment, I never worked with THP1 machophages, but for murine macrophages I incubate the cells at room temperature with gentle shaking for about 5-10 minutes with culture medium with EDTA and then gently pipete against the flask. Trypsin destroy some surface molecules, preventing surface staining for flow cytometry.

For me both gating looks OK...After gating I would preferably gate for either DAPI or PI negative population..sometime apoptotic and necrotic cells can be devoid off from further gating..

It is better to use test gate to distinguish which gate is better. You could use the specific marker of macrophages and set the specific marker positive cells as test gate and then display these cell in a specific color in the FSC/SSC dot, then you could judge which gate is better.

I agree with the responses so far. I think a tight gate is usually better, but with the second population and the fact that you're working with detached adherent cells you'll need to discriminate first. Do a pilot with one or more counterstains (dead cells, surface markers) to help narrow it down by back-gating. Include at least one marker not specific to your cells, as a negative control. Tryspin and chelation/scraping both unfortunately can damage the cell surface and/or lead to cell death. But you likely (?) want to exclude the damaged cells, and they generally form the "halo" around your population, as well as one or both of the larger and possibly even part of the smaller "tails."

You need to put in a doublet discrimination plot. I think you are having some doublets in your plot. Run a FSC-A vs FSC-H or SSC-A vs SSC-H and gate out cells that fall below the diagonal. Then your analysis will be only on single cells.

CD14 FITC or APC labelled should clean it up nicely I'd have thought

Go for 2. You stand a greater chance of gating debris, if you go in for a wide gate like 1. A tight gate gives better specificity..unless your cell population is scattered!

I would consider more or less everything above 60K (FSC) as THP1. We are talking about a cell line here so there should be no other cellular contaminant that a morphological gating should discriminate. I think you are notably losing 3 to 5% of relevant events that are stuck on the axis. You may want to decrease slightly your PMTs (FSC) so that your cells appear less scattered.

I think from your scatter plots you provided the second plot (67%) looks much better. But also it seems that your Voltage and gains a very high such that you are loosing out more cells out of the FSC axis. Again you will need to do dead cell stains to discriminate them out and make sure you are only working with live cells.

I agree with Qingqi; if you really want to know, define your cells of interest and see where they fall back in the FSC-A v SSC-A plot.

I gravitate toward tight gates when analyzing flow. My reasoning is statistical; outliers don't represent the mean, and unless you are looking for an esoteric 1 in 10^6 CTL subset, you will want a mean accurately representing your population of interest (human macrophages). In my experience, tighter gating also cleans up subsequent plots. I would even gate tighter on your tight gate at the top-left. Another thing to consider is: In the end, how many events in your target population do you stand to gain from an extra 3.8% total events? Simple experiment; name 3 populations (tight gate, loose gate and [(loose gate) - (tight gate)]) and run your standard gating on each, then see what your final numbers look like for each population.

I also sugest, as Doris does, a F/SSC-A vs. -H gate to weed out doublets/dead/dying cells. But if you want to use a viability stain, I suggest LiveDead (that may depend on your staining/isolation protocol, though). I also agree with Oscar; your FSC voltage looks high.

In the end, exactly where you gate is up to you and/or the PI. Just make sure you are consistent across your analyses, clean, calibrate and compensate your machine every time you flow and use template gating schemes. Those three things will save you a lot of time and hardship. Good luck.

Hi Abdullah,

If you don't know where your cells of interest are, you should gate as wide as possible in the FSC-A vs SSC-A plot. Keep in mind that the population of dots with high SSC and low FSC are likely the dead cells. And the very high FSC are likely the doublets, triplets, etc.

The next thing you have to do is to make gates for exclusion of dead cells using viability dye and the doublet discrimination by making plot SSC-W vs SSC-H and FSC-W vs FSC-H.

Then you can tighten your gate further by using markers such as antibody F 4/80 for macrophages. At that point if everything goes right, you will find out where exactly the population of your cells are in FSC-A vs SSC-A plot.

So for the first gate (P1) I wouldn't worry too much to make a wide one. Have fun!.

hi,

In general, cancer cell lines (like THP-1) FSC to SSC distribution can be effected by the cell cycle state of the cells since the are constantly dividing. Dead cells and "junk" are usualy found in the lower left wich you excluded from both gates anyway.

I advise to aplly a high and wide gate, excluding the lower left since antibodies can non specificaly attach to dead cells some times.

Human macrophages and monocytes (THP-1) can be labled with CD68 to further assurance.

Abdullah, macrophages (both human and mouse) are notorious for having high autofluorescence. I agree with previous comments on staining for viability to make sure your exclude dead cells from the analysis, and try to work with as low voltage as possible as Oscar Kai suggested above. Your plots will be cleaner. Good luck.

I agree with Ehud. This is a monocytic leukemic cell line so theoretically all live whole cells should be equivalent for analysis. If memory serves these are non-adherent unless further differentiated. Doublets (cells stuck together), dead or dying cells, dividing cells, and pieces of cells are likely causing your FSC vs. SSC scatter. For further analysis you definitely want to exclude dead cells using a live/dead dye like PI and can exclude pieces as Ehud suggests and doublets as Eko suggests. Remember also that there may be a problem with Fc receptors on monocytic cells so may want to pretreat with an Fc blocker to prevent non-specific binding of antibodies.

I'd look at a tighter gate and then also look at total cells. Backgating can help you dice and slice the data. For staining cells that are myeloid, you are better to use red stains to avoid interference with autofluorescence.