Hello,
Have someone used FITC-dextran to measure the phagocytosis of macrophagy? Can you give me a protocol about the working concentration and time?
Why I can not get the result along with my CFU result? But the opposite one....
I have used the FITC-dextran at working concentration about 1mg/ml, for 1 hr. But now I am thinking should I prolong the processing time? As my cell line is RAW264.7, I am wondering the difference between groups is too little to detect. Can someone help me about the phagocytosis test??
Great thanks.
Hi, the best advice would be to do uptake over time test. Start at 30' and go up to 6 hours. The concentration should be fine, but I would add also macrophages without FITC-dextran as your negative control and I would include 30' binding at 4oC to compensate for binding rather than uptake.
Phagocytosis is a fast process, you wills the first uptake around 10min after exposure. As FITC-detran binds to the MMR, which recycles after binding, you really need to include the same set-up at 4C, as already mentioned by Tjerk. There should be NO fluorescence in cells w/o FITC-Dextran (but again, you need both, cells at 4C and 37C), and then do the same with FITC-Dextran exposed cells. The cells at 4C will also take up/bind FITC-dextran, but to a far lower extend than at 37C.
You cannot study phagocytosis with dextran because phagocytosis involves (by definitione) endocytosis of particles which are much larger than even high molecular weight dextran.
Dextran is used for pinocytosis not phagocytosis. For phagocytosis fluorescent latex beads are used
http://www.ncbi.nlm.nih.gov/pubmed/6630865
Check out some publications from our lab and you will find what you need. https://www.researchgate.net/profile/David_Knecht/publications
I would recommend using TRITC-dextran as it is pH insensitive whereas FITC-dextran is pH sensitive. Also load your cells for 2.5 - 3 hr instead of 1 hour. Let me know if you need any further help after going over the papers.
- Gaurav.
FITC-Dextran is used for fluid-phase pinocytosis.
Protocol:
http://digitus.itk.ppke.hu/~cseri/edu/Books/Cell%20Biology/Volume%202/Part%20A/Chapter21.pdf
To make clear how dextran is taken up (phagocytosis, FPE or receptor-mediated endocytosis) you can take a look at my recent review: https://www.researchgate.net/publication/264799444_Targeting_the_C-type_Lectins-Mediated_Host-Pathogen_Interactions_with_Dextran
Article Targeting the C-type Lectins-Mediated Host-Pathogen Interact...
By the way, RAW264.7 should not be expressing mannose receptors that is used by macrophages for dextran uptake.
as we all know there are two sub-types of macrophages (M1,M2), im intending to use dextran uptake assay as a functional assay that may help me to test wether the cells have more M2 or M1 function . which subtype do more endocytosis of dextran M1 or M2 ??
thanks :)
Mannose receptor is one of the M2 macrophages features (for example, read http://www.jimmunol.org/content/177/10/7303.full), so they should be taking dextran via mannose receptor-dependent endocytosis much more effectively.
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