Question

Design a semester long project using RAW cells or mouse macrophages with protocols ranging from counting cells to using flow cytometry with the use of fluorescent microspheres. Other protocols include NO measurement production by Greiss Reaction, detection of apoptosis, western blot. Can use some mice for peritoneal injections with CD11b PE. What I’m trying to say is, if you can design it I can prob execute it.

Answer 1

Cell lines, such as RAW cells or mouse macrophages are highly useful in vitro model for conducting various studies. These cells can be easily grown in laboratory to conduct the desired experiments. Following set of experiments can be designed using these cell for a time period of nearly 4-5 months, where the primary target is to evaluate the following:

1. Experimental initiation dedicated to establishment of these cell lines in the laboratory.
2. Experimental set up to investigate the effect of different doses of CD11b PE on the viability of RAW cells. In other words, different concentrations of this compound can be tested for different time periods and hence the most effective dose of CD11b PE which imparts maximum inter-cellular communication with minimum cell death can be chosen for conducting further experiments.
3. Experiment dedicated to investigate the effect of CD11b PE supplementation on different surface proteins on the RAW cells using immunofluorescence staining by fluorescence microscopy or cell counting using FACS.
4. Evaluation of cellular redox mediator and vasodilator i.e. nitric oxide (NO) by Greiss reaction.
5. Detection of apoptosis by either TUNEL assay, annexin-V binding assay, caspase-activation assay or excessive leakage of ROS.
6. Detection of various markers for investigating cellular markers of integrity, inter-cellular communication and apoptosis by western blotting in cell-membrane proteins, cytosolic proteins, nuclear proteins or organeller protein fractions. This can be performed by using specific cell-lysis buffers.
7. Finally, these experiments can be validated by performing in vivo studies where naive mice might be injected with CD11b PE intraperitoneally and the intestine might be extracted for similar experimental investigations as performed in vitro.