I am asking this question because I am working on my experiments with OVA alexa fluor 488 immune complexes and I only want these complexes to enter through the fc gamma receptors of BMDCs and not leave any free eggs to bind or enter through the mannose receptor.
Currently I do not have the mannan reagent which is known to block this receptor, so I am looking for an alternative with the use of non-fluorescent ova, but I do not know at what concentration I need to pre-incubate the BMDCs with non-fluorescent ova and your protocol, and then add the immunocomplex so that as there is no mannose receptor on the surface, it only enters through the gamma Fc receptors.
Thank you.
To block the mannose receptor (MR) in BMDCs without using mannan, you can try the following alternatives:
-Non-fluorescent OVA pre-incubation: Pre-incubate the BMDCs with non-fluorescent OVA at a concentration that effectively blocks the MR. The optimal concentration may vary depending on the specific experimental conditions and the affinity of the MR for OVA. You can start with a range of concentrations (e.g., 10-100 µg/mL) and optimize based on the desired level of MR blocking and the efficiency of Fcγ receptor-mediated uptake of the immune complexes.
- Optimize incubation time: Determine the optimal pre-incubation time for the non-fluorescent OVA by monitoring the surface expression of the MR using flow cytometry. The incubation time may vary depending on the kinetics of MR internalization and recycling.
- Use a control group: Include a control group in your experiment where the BMDCs are not pre-incubated with non-fluorescent OVA. This will allow you to compare the uptake of the immune complexes between the MR-blocked and non-MR-blocked conditions.
- Monitor MR expression: Throughout your experiment, monitor the surface expression of the MR using flow cytometry to ensure that the blocking agents are effectively reducing MR-mediated uptake. If necessary, adjust the concentration or incubation time of the blocking agents to maintain consistent MR blocking.
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