Sigma sells FITC-conjugated LPS. You can stain your cells in the presence of this LPS-FITC, but beware, you must have serum present in your staining system, which will provide the LPS binding protein (LBP). Stain for 30 minutes on ice for cell surface and 30-60 minutes at 37C for internalization. You may need to play with the LPS concentration and timing.
Miltenyi has a very nice isolation method. It's simple, label your DC with CD11c-FITC or CD11c-PE and isolate them with anti-FITC or anti-PE conjugated beads. Works very well (>90% purity) for spleen and lymph node. We are still working out the bugs with other tissues and thats due to the debris from organs like lung and liver.
You are most likely over trypsinizing your cells and they are dying. Try using 0.5X trypsin-EDTA and they won't die or be very sluggish. Feed when the media starts to change. Once a week may not be good.
For DC, you can use CD205 (DEC205). You can also use CD11c. For macrophages we typically use F4/80. If I remember correctly, you can double stain with CD14 and F4/80 for monocytes (hi CD14) and macrophages (low CD14). There is a great deal published in the area and you may want to do a PubMed search. The markers you chose will only identify subsets of each cell type.
Hi, I don't know if this is helpful but I also tried both semi-dry and wet transfers only because I wasn't getting a good transfer and because I had really crappy (only available) antibodies for detection. If you aren't getting a good wet transfer adjust your methanol from 200 ml/1000 ml to 150 ml/1000 ml without altering your glycine. That was the trick that allowed mt to get the best wet transfer and was superior to the semi-dry. Hopefully, I will complete the manuscript and get it published soon.
I would greatly appreciate assistance in running mouse platelets on the flow cytometer. Although I am able to identify my platelets by CD61 staining, I am still having difficulty with identifying platelets and platelet derived microparticles. Does anyone do this kind of work, and if so, would they be able to assist me in setting my parameters? I found that the platelets and microparticle populations are best identified when both SSC and FSC are set on log scale and the gain is set on e01. Even with these settings, I am picking up a great deal of non-specific events even when I try to set the flow using purified washed resting platelets. Thanks.