A Protocol for Revealing Oral Neutrophil Heterogeneity by Single-Cell Immune Profiling in Human Saliva
Updated August 30, 2022Neutrophils are the most abundant white blood cells in the human body responsible for fighting viral, bacterial and fungi infections. Out of the 100 billion neutrophils produced daily, it is estimated that 10 % of these cells end up in oral biofluids. Because saliva is a fluid accessible through non-invasive techniques, it is an optimal source of cells and molecule surveillance in health and disease. While neutrophils are abundant in saliva, scientific advancements in neutrophil biology have been hampered likely due to their short life span, inability to divide once terminally differentiated, sensitivity to physical stress, and low RNA content. Here, we devise a protocol aiming to understand neutrophil heterogeneity by improving isolation methods, single-cell RNA extraction, sequencing and bioinformatic pipelines. Advanced flow cytometry 3D analysis, and machine learning validated our gating system model, by including positive neutrophil markers and excluding other immune cells and uncovered neutrophil heterogeneity. Considering specific cell markers, unique mitochondrial content, stringent and less stringent filtering strategies, our transcriptome single cell findings unraveled novel neutrophil subpopulations. Collectively, this methodology accelerates the discovery of salivary immune landscapes, with the promise of improving the understanding of diversification mechanisms, clinical diagnostics in health and disease, and guide targeted therapies.
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Atlas
Analysis Portals
NoneProject Label
PRJNA640427_human_neutrophilsSpecies
Homo sapiens
Sample Type
specimens
Anatomical Entity
oral cavity
Organ Part
saliva
Selected Cell Types
neutrophil
Disease Status (Specimen)
normal
Disease Status (Donor)
normal
Development Stage
human adult stage
Library Construction Method
Smart-seq2
Nucleic Acid Source
single cell
Paired End
trueFile Format
Cell Count Estimate
1.1kDonor Count
3