In pneumonia, lungs harbor a variety of resident and recruited cells participating
in immune responses as well as maintaining organ functionality and physiology under
stress. Quality and completeness of a potential murine pneumonia atlas depend on the
quality and diversity of the single cell suspension input later subjected to single
cell RNA sequencing (scRNAseq). We aimed to isolate as many different cell types with
the highest viability and lowest stress response possible. Herewith, we adapted available
protocols initially established to isolate fragile alveolar epithelial cells, to allow
simultaneous isolation of endothelial cells and leukocytes. Dissociation of lung cells
from their matrix into a single cell suspension was achieved by enzymatic digestion
and mechanical processing, followed by dead cell removal via magnetic cell sorting.
The obtained single cell suspensions were analyzed by flow cytometry for presence
of doublets and dead cells in the following cellular populations: Alveolar macrophages,
neutrophils, inflammatory macrophages/monocytes, eosinophils, vascular and lymphatic
endothelial cells, type 1 and type 2 alveolar epithelial cells, and bronchial epithelial
cells. The viability of all cellular populations analyzed were above the mandatory
80% required for single cell partitioning and barcoding, while doublet rates were
in the expected range for the individual cell populations. Single cell suspension
quality allowed for Gel beads in EMulsion (GEM) generation and barcoding, followed
by cDNA amplification and library generation. Final conclusions with regards to the
matrix dissociation steps – necessary to isolate single endothelial and epithelial
cells – inducing stress responses that ultimately overwrite the cellsʼ pneumonia signature
will be drawn following attentive analyses of sequences libraries.
