It's really easy.  Just follow the protocol.  I can't imagine there's a need to develop it with a cell line first.  If you do it by the book, nothing will go wrong. 

The only "trick" we came across is that you want to check on the microscope as well, to make sure that they don't just "quantitatively" glow, but also "qualitatively" end up in lysosomes.  On the flow by itself, you can't easily distinguish bound from internalized particles.

I would not recommend the plate reader.  It cannot distinguish between endocytosed, bound and unbound particles.  Whatever it reads needs to be validated by microscopy and flow no matter what.  Plate reader has no advantage (?), so you might as well only use microscopy and flow. 

Hi Aleks - Vybrant is not the best kit for your purposes. You will be better off with a pH-sensitive dye that fluoresces only when phagocytosed:

pHrodo® Green E. coli BioParticles® Phagocytosis Kit for Flow Cytometry

https://www.thermofisher.com/order/catalog/product/P35381?ICID=search-product

They may have other dye/bacteria combinations as well.

Hi Aleks,

The advantage of the Vybrant in combination with the plate reader is that you can quench the extracellular signal of unbound and bound, but not internalized particles (that's why they provide the trypan blue). Although I would recommend checking that this is true under the microscope, you wouldn’t need to verify bound vs. internalized particles in each and every experiment. If you decide to use this assay, I would also play around with the concentration of the quencher solution. If you’re unlucky and it’s too concentrated, you might get quenching inside the cell. However, I agree with Anton that there are better solutions, like the pH sensitive particles. I started using the Vybrant, but after a while switched over to the fluorescent particles from life technologies (pH sensitive and insensitive). Using those also gives you the freedom to use a different channel on the FACS and play around with the multiplicity of your particles, brightness and spillover more easily.

What you do depends on what you want to measure. For instance, the dose of bacteria that you give them (i.e.MOI 1,10,100,etc.) will show you differences in the populations of cells that respond (i.e. phagocytose). Increasing the MOI will increase the % of cells responding up until you reach a saturating dose. This is not necessarily due to differences in binding of the bacteria by the cells.

For adherent vs internalized, trypan blue will help quench the extracellular fluorescence, as Sarah suggested. It should be added immediately before acquisition. It will fluoresce itself around 610nm into the infrared. See my manuscript for details to work with this.

pH sensitive particles must be used carefully. pHrodo green and red are both fluorescent at neutral pH, but their fluorescence increases with decreasing pH. Therefore, they do not tell whether increases of fluorescence are due to decreases in phagosomal pH, or increased amount of internalization albeit at less acidic pH (i.e. 10bacteria @pH 4 MFI=1,000, 100 bacteria @pH 6 MFI=1,000).

pH sensitive fluorphores must be used with a calibrator (i.e. pH insensitive fluorophore such as Alexafluor-350). Please see manuscripts below

http://www.ncbi.nlm.nih.gov/pubmed/26371253

http://www.ncbi.nlm.nih.gov/pubmed/19937952

http://www.ncbi.nlm.nih.gov/pubmed/15825183

Do you not have access to a luminometer, which is the most appropriate instrument for measuring phagocytic activity, using a bioluminescent indicator such as luminol. It seems once folk have a flow cytometer they try to use it to measure everything - when better alternative technologies may be available.

George,

How would using a luminometer be the most appropriate approach? Luminol  will indicate the production of reactive oxygen species, which can happen  in both intracellular and extracellular compartments. Phagocytosis does not need to occur to have production of ROS (e.g. fMLP stimulation, Pattern Recognition receptor engagement). Luminol is in no way specific for phagocytosis.

John, thank you. I'm glad it worked for you, hearing this brings me some reassurance. I used a modified version of the recommended protocol suggested by this protocol book I have for neutrophil work. It didn't work for me, so I will definitely try the original protocol next. I hope it will work fine

Anton, thank you for your suggestion. I will have to keep it in mind as I go further in the project. Unfortunately, this is what I have on hand and I am going to have to try it out first and see where it gets me. I will certainly look into the other assay

Sarah and Evan, you certainly have some very good suggestions. I'm so thankful for your input. As I mentioned to Anton, right now I have to work with what I have on hands, and just see where it gets me right now. You have mentioned some aspects that I haven't considered Its going to be very helpful. Thank you for the details and your explanations. 

Evan, what I basically want to look at, is if there is any difference in the rate of phagocytosis for neutrophils in different age groups. 

George, yes, we have the equipment. I think down the road we are going to use some of these other assays as well. However, for right now, I would like to work with this one and see if i can make it work for us

Aleks,

I have a suggestion for determining differences in PMN phagocytosis across age groups.

Give 2 different doses of bacteria to each sample; a high and a low dose.

The high dose would tell you the capacity of the the PMNs to phagocytose. The low dose would tell you what proportion of cells are primed/active enough to respond quickly to a more physiologically relevant dose. 

Determine the high and low dose through simple titrations. The high dose (saturating ) will give the highest MFI and any dose above that will not increase the MFI. The low dose is a bit more arbitrary, but it will probably be 20-100x less bacteria.

This approach might show something interesting such as Young PMNs phagocytose much more at low doses, as compared to PMNs from elderly, however, the total capacity (high dose)of PMNs to phagocytose is equivalent across all age groups. That is a for instance, you might find completely different results.