I am working on an antigen presentation assay using thp-1-derived macrophages and OVA as model antigen. For differentiation I add PMA for 48 hrs to 50000 cells per well (96 well plate) in RPMI containing 10% FCS. Then the cells rest for 24 hrs in PMA-free medium. After washing the cells 2x with DPBS, I add OVA at a final conc. of 30 micromolar in RPMI containing 10% human serum. The cells are incubated for 2 hrs at 37 °C. Then I wash the cells with DPBS, fix the cells with 2% PFA for 1 h at RT, wash the cells again 2x with DPBS again and store them at 4°C. I plan to detect the amount of expressed MHCII complexes with bound OVA fragments at the surface using Alexa fluor 647- coupled primary HLA-DR antibody. For this, I block the remaining space in the well with 5% BSA for 1 h at RT. Finally, I add the antibody at a final conc. of 5 microgram/ml in DPBS containing 1% BSA and 0.02% sodium azide. I incubate the plate over night at 4°C, then wash the plate (over the sink) 5x with DPBS and measure fluorescence using a fluorophotometer.
- So far I couldn't detect any significant fluorescence signals. Can you tell me which steps should be optimised?
- And do you think that additional opsonisation with IgG is needed? We already incubate the cells in the presence of human serum - so, in our opinion, endocytosis should work....
- Do you think that we should extend the incubation time with the antigen?
I found several problems with your experiment
I hope this information helps you to plan the experiment better. Good luck
Ramachandramouli Budida, thank you for your answer.
1.) I do not necessarily need to differentiate MHC class II with OVA from other MHC class II complexes - I think it would be (for now) sufficient for us to show that after incubation of the cells with OVA, we can detect an increased number of MHC class II complexes on the membrane compared to cells which were incubated in the absence of OVA.
2.) Yes, this was done first - but I was afraid of the possibility that the cells may degrade the newly exposed MHC class II-OVA-complexes during the incubation time with the antibody (O/N at 4°). Maybe it this case I should incubate the cells with the antibody just for 1 h at 37° and then fix the cells?
3.) As "negative controls" I use cells which were incubated in the absence of OVA. In this case, we should just observe the basal expression of MHC class II complexes. This can be considered as our background fluorescence. Besides, we also have unstained controls to ensure that the observed fluorescence is not autofluorescence of cells.
Dear Agnes,
You need to understand several theoretical and practical concepts before starting with your experiments.
For more details on activation of macrophages, you can refer to the attached article. You need to do a lot of background work to design this kind of experiments. Good luck
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