I have done the phagocytosis experiment following the protocol of the research article Giamis et. al. 1992 exactly. I had taken 10^5 macrophages and 10^7 yeast cells per well for a 24 well plate. I had used round cover slips for reaching 100x magnification on an inverted microscope. I am confused as someone said that there are no visible macrophages in my images even in macrophage only image. Please suggest and enlighten me on this issue. Thanks in advance.
I am attaching herewith an image containing only macrophages and one image with both yeast and macrophages.
Images are at 100x magnification and taken at under inverted microscope.
Either in the image what you are saying "Only macrophages", or in the one with yeast cells, there are no macrophages. All the seeded macrophages that were seeded would have undergone death either due to contamination or by any other reason. You haven't provided any info. about what that pink color is. More over, info. such as 1) what macrophages are they, 2) how are they seeded, 3) treated or untreated is missing in your question. A detailed question will be helpful for better understanding and guiding you with a better protocol
Okay. I have seeded the cells and kept for 2 hrs to adhere to the coverslips put in 24 well plate. The cells were murine peritoneal cells. I had treated the cells with Exopolysaccharide (EPS) produced from lactic acid bacteria and then these activated macrophages were incubated with yeast to estimate the phagocytic activity of macrophages. The cells were treated with 1% tannic acid before staining so that only yeast cells that have not been internalized takes the blue color while the yeast cells that have been phagocytosed takes the light pink color so as to distinguish between the phagocytosed cells from the non phagocytosed cells. The pink color is due to Giemsa staining. In this protocol, we had used May Grunwald and Giemsa staining wherein the blue color of yeast is due to May Grunwald stain.
It is nice to have response from you.
The more detailed protocol is given in the research article mentioned in the description of the question.
With an understanding that the protocol was followed exactly as detailed in Giamis et. al. 1992, the possible interpretation for not seeing any macrophages could be due to either low cell binding on the cover slips used leading to loss of cells during staining and washing. Try coating the cover slips with polylysine or soak the cover slips in diluted fcs before using them.
However, macrophages are strong adherent cells by nature. With this in mind, there could be a possible contamination leading to cell death and that is quite evident by your "Macrophage only" image. One question to think is "do you sterilize the coverslips"? Possible contamination on the coverslips may kills the cells. Checking the viability status of these cells by a simple Trypan Blue test before going ahead with phagocytosis assay may help.
As you mentioned that these cells are murine peritoneal cells, with an understanding that you would have isolated these cells by thioglycolate means, looking into your cells isolation protocol may help.
BW
- Badges
- Science method