Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

Abstract

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

Hyperalgesic Effect Evoked by Il-16 and Its Participation in Inflammatory Hypernociception in Mice

“…Samples were incubated at 37ºC for 90 min and gently shaken 1-2 times during incubation. Skin digested samples were collected into 50 ml tubes, being poured through 40 µm cell strainer that was washed with 5 ml of RPMI 1640 supplemented with 5% FBS (Broggi et al 2016). Finally, samples were analyzed using surface and intracellular staining by ow cytometry as previously described (Sordo-Bahamonde et al 2020).…”