Hi, I am trying to label chicken dendritic cells using Dec205 antibody for flow cytometry analysis. I make the DCs from bone marrow cells using IL4 and GMCSF. I also induce the cells with 200 ng/ul LPS for 24 hours before labelling for flow cytometry. Inducing with LPS allows the expression of Dec205 receptors on the cell surface. When I observe the cells under the microscope I see dendritic cells like morphology. However, I have not been able to label the DCs with Dec205 antibody. In literature, they have shown that Dec205 is one of the prominent dendritic cell marker in chicken. I have also not been able to label DCs with MHCII antibody. Before labelling I trypsinise the cells (1xtrypsin) for detaching it from the 6 well plate and gently scrap the cells. I was wondering if trypsinising the cells is bad for the DCs? Any advice and suggestions will be very much appreciated.
Yes trypsinising is bad for most surface marker studies not only just in DCs. So like Iryna has suggested you can leave the cells for a few minutes in a gentle detachment solution like 0.5 - 2mM EDTA in Ca & Mg free DPBS after washing the cells with DPBS twice. The concentration of EDTA and incubation time may vary depending upon the cell line. I mostly use 0.5 mM. Although this works for most cell lines some cells such as HepG2 may not come off. In such cases you can use gentler enzyme mixtures such as Accutase.
Make sure you wash the cells properly after harvesting to remove the EDTA which may interfere with some assays.
Dear Angita,
Trypsinization is not recommended for surface marker staining in flow cytometry as this harms the surface antigens. I agree with Ashutosh Acharya and Iryna Romanivna Horak in detachment process they described. You need to standardise the concentration as per your need.
Good luck !
Thank you for the suggestions. Do I incubate the cells with PBS+EDTA at room temperature or 4C? And is 10 minutes enough?
Dear Angita,
10 minutes should be fine but again you need to standardise. While you are doing your cell detachment step, mild chilling should do good, so you can incubate the cells in PBS+EDTA at 4C.
Good luck!
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