Hello, I am doing an assay on neutrophil phagocytosis using C. albicans. Let say I have two sets of experiments: the untreated and the treated sets. I would like to compare the phagocytosis ability of neutrophils in both sets by counting the number of yeast that has been engulfed by neutrophils. How do I count neutrophils? On a slide, how many neutrophils should I count? Do I count only neutrophils that have at least one Candida inside or do I count neutrophils without Candida too? What about neutrophils that have Candida in contact but not completely engulfed, do I count those neutrophils too? Thank you very much in advanced.
Dear Decha, hello. First of all you have to know the neutrophil/ Candida ratio before co-incubation and use the same ratio both for control and for experiment. To count of neutrophils should take a small quality of cells, stain them by Tuerk or other nuclear dye, placed in a special chamber for blood cells counts and count the number of neutrophils. Then you are diluted to the desired working concentration. You can simply count the number of neutrophils in the flow cytometer. Candida can be calculated or under a microscope (but it is very bad for the eyes method!) or by optical density using a spectrophotometer. Then Candida are diluted to a working concentration. After than you incubated Neutrophil+Candida, prepare the smear and for calculate the phagocytic index you view 100 neutrophils and calculated: 1. The number of cells with Candida, 2. the number of cells without Candida, 3. the number of cells with "phagocytosis ripped off" - usually on the smear they more dark. It is a died neutrophils as a result of phagocytosis. True, this phenomenon is more common for Staphylococcus, for Candida rarely.
I used to do this quite frequently. However, I'm not clear on what you're trying to answer - the effectiveness of neutrophils in phagocytosis or in Candida albicans to survive.
If it's the first issue (neutrophil activity) then time-lapse microphotography is a good way to observe this. I agree with the issues raised by Dr. Pleskova about knowing the ratio of neutrophils to yeast ahead of time. I found that a hemacytometer works well for counting both neutrophils and yeast, and it's low cost. You could count both on a flow cytometer if that's available to you. This would give you a more accurate count of a higher number of cells. It also has the advantage of "arresting" the cells at a given time point because of the fixing done prior to loading on the flow cytometer.
If you want to assess the survival of Candida albicans in the presence of neutrophils then performing an MTT assay on a plate reader is quite easy. The process is to co-incubate the neutrophils and yeast for however long you want, add water for 10-15 min to lyse the PMNs, centrifuge, resuspend yeast pellet in PBS, then add MTT, incubate for 30-60 min. Depending on how many viable Candida cells are left over there will be a color change with the MTT, yellow to purple. It's easy to quantify this on a plate reader. Again, need to pay attention to the ratio of PMNs:yeast and making sure that if comparing different ratios that the starting yeast amount is constant between wells. Otherwise you could get misleading results.
Dear Dr. Pleskova and Dr. Lyon,
Thank you very much for your answer. Let me explain what I was doing.
1. I counted neutrophils with a hemacytometer. The neutrophils were strained with trypan blue.
2. I determined the number of live yeast cells by plating at a certain OD600. With that I got an idea about the cell density and the estimated number of cell.
it is quite difficult to get an accurate number of living yeast cells from this technique but this is the best I can do. The number I get is more of the estimate. Then, I incubated neutrophils with Candida in the ratio 1:1. After 60 min of incubation, I diluted my sample with PBS and then water (10 min room temp incubation with water to lyse neutrophils). Then, I plated on PDB plates and determine the number of Candida that survive after incubation with neutrophils.
To determine whether a decrease in the number of living yeast is due to phagocytosis, I spreaded 10 ul sample on a slide for staining with Wright-Eosin Methylene Blue stain. But I don't know what is the standard practice or method for determining phagocytosis of neutrophils. That's why I asked that question.
Dr. Pleskova: i think your #3 is tricky and I think it take much of the skills and practices, right. Say if I got #1-#3, how do I report this data?
Dr. Lyon: I like your idea of using MTT to determine the number of surviving yeast. I'll try it.
I'd like to thank both of you for your help. I will try as you suggested.
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