I have heard of Neutral Red uptake assay for macrophages; however, I've never tried it, and have no idea on the particle size. I would be interested to know what techniques are recommended for this work.

It really depends what you want to study. Lot of people used PS fluorescent beads, E.coli/zymosan will activate other cell membrane receptors (TLRs) and then internalization. If you want to study a specific pathway I would recomend you specific antibodies (such those for early and late endosomes) to track the route of internalization.

However if you want a positive control for phagocytic activity you may try some sub-micron particles with low toxicity (PS as I said, partly titania dioxide, amorphous silica...) along with E.coli LPS.

Maurizio

Thanks for the info Elizabeth and Maurizio. I am not interested in a specific pathway, I am just looking for a positive control to study phagocytosis in macrophages.

Dear Pia. Well, in that case - either latex beads or zymosan particle are an easy target as these can be seen by light-microscopy as well. If you want more sophisticated methods, any dye-expressing E.coli will do. Check Molecular Probes (now sold through Invitrogen) for this. Problem is you may need to quech colour a bit if you do it by flow/confocal. Otherwise - latex-/zymosan-beads are the simplest solution. Hope that helps?

What about FITC-labled dextran, which is a standard particle for such an assay? Or latex beads labled with fluorochrome-conjugated antibodies. These latex beads are available in many different sizes.

Bastian

Hi Pia. I would recommend E. coli B 1001 for analysis. It is simple, non pathogenic and you can prepare, by standard methods, a lot of bacteria FITC and conserve it at -70 degrees. Alternatively, Streptococus or Salmenella may be good choices, however, they are more difficult to manage.

good luck

Maurizio's suggestions are good. It depends how you want to/what resources you have to determine uptake. Fluorescent beads are the most usual method-either by flow cytometry or microscopy. If resources are limited or there is no access to flow cytometry simple microscopy of uptake of latex microbeads (most commonly used), killed bacterial cells/yeast cells or even carbon partculates have been used. The use of bacteria/yeast can of course stimulate cells without uptake so the use of neutral particles is best as a straightforward assay.

I agree to Maurizio. It really depends on what you want to study. Many phagocytosis assays use IgG-coupled latex beads which are taken up after recognition by Fc receptors. These kits will allow you to analyze IgG.mediated and Fc receptor-dependent phagocytosis. This phagocytic pathway is different to that used by macrophages to engulf dead (apoptotic) cells, which are recognized by phosphatidylserine residues located on the outer leaflet of the membrane of apoptotic cells. Macrophages use different receptors to detect this (PS-receptor or integrins via adaptor proteins such as Mfge8).

So, as mentioned initially, the assay really depends on what phagocytic pathway you study.

Regards, Björn.

you could use pHrodo E. coli or S. aureus which only fluoresce once fully ingested into mature phagosome….

http://www.lifetechnologies.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/phrodo-indicators.html

Dear Pia,

antibody-opsonized Escherichia coli-Alexa Fluor 488 (Molecular Probes #E-13231) are straightforward particles. They target Fc-receptors and therefore are appropriate to study phagocytosis in macrophages. Thirty minutes to 1 hour at 37°C gives a good signal. After washing make sure to quench fluorescence of extracellular bacteria with 0.4% trypan blue.

Best of luck for your experiments

Loems

Dear Pia

I have questions for al the responders ,has any one worked on macrophages? if so then the answers are applicable . Since I have worked on it as my PHD ,therefore, I can answer the questions. Pia please search on goggle all my papers author named

Pratibha Gupta and Ajit Sodhi on macrophage, activation .You will find all my papers , information and methods also so you do not need to search more as you can also look for cross reference from the papers. Pl contact me personally at [email protected] .Thanks Dr. Gupta

As a Comparative Immunologist, I must also add that host species of macrophage or other phagocytes also has profound effects. For example, bovine monocytes adhere poorly to glass beads and fMet-Leu-Phe is usually not chemotactic for bovine neutrophils (which is normally the case for rodents and humans). Macrophages and other leukocytes in lower vertebrates also respond in very totally different fashion from conventional models, depending on the particle and sometimes, even on which fluorescent dye is used (if one is doing a fluorescent assay). I've found defibrinated sheep erythrocytes coated with a subhemolytic amount of mammalian immunoglobulins against erythrocyte stroma and cultured 30-60 minutes at 37 C works well for most mammalian species. For lower vertebrates, I'd recommend a particle that would fix complement by the lectin pathway. This could be zymosan or E. coli, as several others have suggested or it could be a member of the typical microbiota of whichever species your macrophages come from.

JP Caruso, PhD

FITC-labelled zymosan.

ttp://www.jimmunol.org/content/182/2/1146.long

I used the opsonized by the pool of native serum labeled C14 Staph. aureus to study the 24-hour dynamics of phagocytosis by blood mononuclear phagocytes, as well as the FITC-labeled aggregated immunoglobulins, the last objects might being more adequate for the rheumatoid arthritis, which I study. When the task appeared to investigate the intensity of the exocytosis of lysosomal enzymes from the monocytes using fluorescence labeled substrate for lysosomal glucuronidase , I had to take the opsonized zymosan as the other objects of phagocytosis had cross- fluorescence with the labled substrate.

Actually, what do you mean under the term "phagocytosis"? The whole process from the reception of the objects up to the release of the digested scraps from the cells? Or only the injestion of the particles by cells?

Study of ROS generation - it is another story!

This might also help:

https://www.bangslabs.com/applications/phagocytosis/fluorescent_polymer

I've recently used these particles with good results for confocal and imaris 3D imaging of engulft particles within primary macrophages. It is moreover possible to quantify the phagocytic process afterwards by flow cytometry.

I suggest you use europium infused micron size latex particles coated with Immunoglobulins targets. I had great success I'm similar situation. Uptake of particles by macrophages in many natural situation is through Fc receptor cooperativity of binding is criticalI. High density of IGg is a trigger

I also agree with many of the previous commenters and have seen a number studies which use E. Coli or Zymosan. Activation of the surface receptors tends to improve phagocytosis, so if you are going to use beads considering opsonization. We have had some success using fluorescent polystyrene microspheres from Bangs Laboratories, which have the added benefit of being bright and easy to see by microscopy and FACS.

Yellow-green fluorescent Microspheres from Molecular Probes (well, now it's Life Technologies) are easy to use and sensitive. They come in different sizes: e.g. FluoSpheres® Carboxylate-Modified, 0.1 µm

Depending on what phagocytic receptor you are looking at:

If CR3 or FcR, then I would use latex beads or sheep red blood cells - both of which are inert targets... just opsonise with C5 deficient serum (only C3bi deposited) OR IgG.

If you wish to understand dectin-1 or -2 receptors, then zymosans is ideal (Herre J et al, 2004, Blood).

If mannose- or scavenger- receptors, then you might have a problem trying to distinguish these two... but others on this board might help.

Hope this is useful.

Regular baking yeast is a simple way to visualize phagocytosis. Boil and wash by centrifugation.

Dear Pia,

Zymosan and E. coli are suitable, moreover you can also utilze 0.1 micrometer fluorescent latex spherules to study phagocytosis of macrophages in vitro. You can add these fluorescent beads in a 1000:1 ratio with an incubation at 37°C for 4h.

Regards,

Antonella

If one wants to measure the intrinsic phagocytic activity of macrophage and its dependence on physiological or immunological factors, regulatory mediators etc, but irrespective of this cell's possible damage and/or activation by the phagocytized objects themselves the latter should be as biologically inert as possible. Therefore neither any bacteria, nor zymosan, nor amorphous silica particles (in fact, much more cytotoxic for macrophages compared with quartz particles) are a good choice. My own experience makes me vote for 1 mcm polystyrene latex beads.

They are two different foreign particles which can give different responses. For a routine assay, I usually use baker's yeast: it is easy to detect under the optic microscope.

In my eyes, GFP or dsRED expressing E.coli (or other bacteria/funghi) are suitable for FACS based phagocytosis assays. It depends which receptor/ligand interaction you are going to examine....

The best phagocytic particle to assay phagocytic activity of macrophage is

the soluion of carbon ink , the mixture consisted of black carbon ink 3ml, saline 4ml and 3% gelatine solution 4ml.

This is really a very broad question and depends on the motive for performing your phagocytosis assays; many of the responses are helpful, especially Jensen's. If your interest in the macrophage immune response to pathogens then use heat killed pathogens, eg E.coli or Mycobacteria, bearing in mind that they will engage several different receptors. E.coli labelled with a pH-sensitive fluorophore such as pHRODO is probably the best target for flow-based phagocytosis assays and we have used them to determine phagocytosis by monocytes in patient whole blood. You can also opsonise the pathogen with immune serum or complement if required. If you are interested in looking at specific receptor-mediated mechanisms we have used red blood cells opsonised with antibody or complement components to determine what effect HIV infection of macrophages has on specific receptor signaling pathways. Red blood cells also have the advantage that internalised targets can easily and sensitively be quantified in high throughput plate assays by measuring the hemoglobin ingested. Latex beads, are the classic target and can be coated with BSA/antiBSA IgG to measure FcgammaR mediated ingestion. The size of the particle is important as it will determine the mechanism of uptake and the signaling molecules required, as shown in Joel Swansons lab many years ago. Beads >1um are usually used for phagocytosis and we have used fluorescent 3um beads successfully.

Dear Pia, The easiest phagocytosis method is with carbonyl iron - an old method, not included in the methods' books of the last decade. Using a light-microscope one can count number of particles taken by cells with certain morphology.

We routinely use the pHrodo particles from Life Technologies. I believe they are one of the better products because the particles only fluoresce once phagocytosed (due to change in pH). They are easy to use and eliminate the problem of particles sticking to the cell being included in your analysis.

Dear Pia, latex beads are best option as you can coat them with different Ligands very easily and also can purify phagosoms ( with >95% purity) by single step sucrose gradient. Moreover they are available in different sizes and different colors but fluorescent labelled beads are relatively more expensive. Your question do not contain enough information for specific suggestions. If you can explain your intended use in more detail we might able to help with specific suggestions.

I would like to point out an important fundamental feature of phagocytosis. When you seal a flap of envelope, whether it is an adhesive envelope or whether it needs to starch the flap, you must press the flap against the envelope. Like sealing envelope, physical impact is essential for particles to be recognized by cells. In case of phagocytes such as neutrophils and macrophages, various opsonins play the role of starch. Only a slight response (including phagocytic response) of these cells can be observed even when more than 1000 bacteria (opsonized or not) per single cell are added, if bacteria are non-motile like Shigella and motility mutants of Salmonella, E. coli and Pseudomonas. If non-motile bacteria are centrifuged together with a monolayer culture of macrophages, the number of bacteria ingested per cell increase dramatically. The minimum centrifugal force required for maximal response, is 5 and 2 xg in case of non-motile bacteria and zymosan particles, respectively. The velocity of bacteria calculated from the g value is about 10 times lower than the velocity of movement of motile bacteria. That means physical impact caused by bacterial motility is enough to induce a high response. Therefore、if bacteria are injected into mouse peritoneal cavity and macrophages are washed out, number of motile bacteria ingested per cell are much higher than that of non-motile bacteria. We have reported these findings long time ago (2 papers in Infection and Immunity, 32: 1242-1248, 1981 and 38: 865-870, 1982). Physical impact seems to help infection of bacteria such as Vibrio cholerae and efficient gene transfer using centrifuged virus particles (usually virus particles are present in media as aggregate).

Dear Pia, Working with Zymosan is very easy if you can use a chemiluminescence system. In this case luminol excites light induced by the respiratory burst.

For flow cytometry and visualization in the fluorescence microscope, you can use fluorolabeled E.coli provided by several companies. In this respect, BD offers a phagocytosis kit for flow cytometry. Above all, it is important to opsonize the respective particles. For this purpose I used freshly reconstituted lyophilized rabbit complement.

I agree with many previous answers that opsonized zymosan is a very good tool to assess phagocytosis process. I attached an unpublished figure showing a macrophage (RAW264) in process of engulfing opsonized zymosan particles. After 12 hr of ingesting zymosan, the autophagic vacoules are visible in these macrophages detected with GFP-LC3 tagg.

Many thanks to all of you for your useful suggestions! :)

Hello pia

we tried zymosan particles with invertebrates haemocytes and mice blood and it worked very effectively for both of them.

in flow cytometry with two color assay, color will chage when the particles come in macrophages. This method will help us to differ the particles in and out of the cells. I can share this literatüre tomorrow

flow cytometry two color assay with ethidium bromid

Saresella M et al. A rapid evaluation of phagocytosis and killing of candida albicans by CD13+ leucocytes J Immunol Methods 1997;210: 227-234

Phcoerythrin tagged latex beads are ideal if you want to visualise the phagocytosis by flow cytometry.

How if we want it from intravascular blood sample look for macrophage activation?

Zymosan (or also baker's yeast is easy to use and detect under teh light microscope and inexpensive. Bacterial cells are much more difficult to be revealed.

Dear Pia. I agree that the technique using the zymosan is very easy to proceed and efficient to study macrophage activity.

Hi Pia,

we are using GFP expressing E.coli and PHrodo E.coli. Within short intervals of infection (1 - 4 h p.i.) GFP (or dsRED expressing E.coli are easy to detect via FACS. You should keep in mind that - utilizing FACS Analysis - it is hard to distinguish between attaching or CD14 binding bacteria and E.coli which are incorporated. Think about a second staining with an LPS specific antibody - "double positive" E.coli (for example GFP-expressing and anti LPS-PE) are outside and single stained ones (GFP-expressing) phagocytosed. Here, the PHrodo E.coli help to get an idea of phagocytic efficency. Also Keep in mind, that E.coli infection causes downregulation of CD14.

For longer infection periods (up to 24 hours) consider the digestion of bacteria and GFP.

Good luck