The effect of enzymatic digestion of tonsillar tissue on the yield and pattern recognition receptors expression level on human dendritic cell subsets

The development of vaccines against tumours are gaining increased attraction and one strategy to increase the efficiency of these vaccines is to target the antigen or drug to cell surface receptors on dendritic cells (DC), such as the C-type lectin receptors (CLR). To this end, knowledge of the expression levels of CLR on DC subsets is important and it has been studied in different tissues and blood, most commonly using flow cytometry. However, these receptors are prone to be affected by the digestion enzymes used to obtain single cell suspension from primary tissue. In this study, we investigated the DC yield as well as the expression of several CLRs on DCs after treatment with two commonly used digestion protocols; a protocol commonly used within the field of DC research (Standard Enzyme protocol) as well as a commercially available kit, compared to a control where no enzymes were applied. Single cell suspension of tonsillar tissue, as well as peripheral blood mononuclear cells (PBMCs), was stained with antibody (Ab) panels and cell surface expression levels of various CLRs was analyzed using flow cytometry. The results demonstrated reduced expression of CLR surface receptors CLEC9a, DEC205, CLECSF14 and Dectin 1 using the Standard Enzyme protocol but not when using the commercial kit. The yield was not affected for CD141+ and CD1c+ DC between the different protocols but a decrease was seen for CD123+ DCs when using the Standard Enzyme protocol in comparison to the Control protocol. This study stresses the importance of selecting the appropriate protocol for the downstream analysis and suggests that previous studies on these CLR should be interpreted in relation to the enzymatic degradation applied.