Fig 1.
Neutrophil purity after isolation using Polymorphprep or negative-selection (magnetic beads).
(A) Percentage of leukocytes in each preparation from each donor. Cells quantified by cell morphology and staining properties using cytospins (calculated from 4 separate fields of view, counting > 100 cells per field per donor). (B) Representative cytospins of neutrophil preparations following Polymorphprep (Poly) or negative selection (Beads) isolation protocols from Donor 1 (top) and Donor 2 (bottom). White arrows highlight non-neutrophil cells. (C) Flow cytometry scatterplots of neutrophil preparations by Polymorphprep (Poly) or negative selection (Beads) isolation protocols. Plotted by green fluorescence (CD16 positive, X-axis) and forward-scatter (Y-axis). Donor 1 (left panels) and Donor 2 (right panels). Numbers shown are percentage of cells in each of the two quadrants shown. (D) Levels of expression of cell surface markers in neutrophils isolated by Polymorphprep (Poly) or by negative selection (Beads). Geometric mean fluorescence (GMF) of CD16 (N = 5), CD15 (N = 3), CD11b (N = 3) and CD64 (N = 4) was measured by flow cytometry and normalised to an appropriate isotype control. Error bars represent SEM.
Fig 2.
Neutrophil yield using density gradient isolation or negative selection.
(A) Neutrophil yield (106/mL) from whole blood isolated by Polymorphprep and negative selection (Beads). Data represents n = 5 paired neutrophil isolations (**p<0.01). (B) Neutrophil yield (106/mL) from whole blood isolated by Ficoll-Paque (Ficoll) and negative selection (Beads). Data represent n = 5 experiments in which neutrophil enrichment from the Ficoll-Paque granulocyte pellet was carried out using negative selection (**p<0.01).
Fig 3.
Correlation between transcriptomes of Polymorphprep and negatively-selected neutrophils.
Gene expression (RPKM) in neutrophils isolated by Polymorphprep or negative selection (Beads) for Donors 1 and 2 following incubation with or without the addition of GM-CSF (5ng/mL) or TNFα (10ng/mL) for 1h. Pearson Correlation co-efficient is shown (p<0.01). The solid line indicates a correlation coefficient of r = 1.0 and the dashed lines indicate a fold difference in expression of 2.
Fig 4.
Expression levels of non-neutrophil genes in neutrophil preparations.
RPKM values for non-neutrophil genes of the antigen targets in the StemCell magnetic bead negative selection isolation kit (A) and (B) non-neutrophil specific genes associated with T and B cells, monocytes, and eosinophils. Neutrophils were either isolated by negative selection (Bead, circle) or by Polymorphprep (Poly, square) from Donor 1 (1) and Donor 2 (2). Neutrophils were treated with 5 ng/mL GM-CSF (shaded grey), 10ng/mL TNFα (shaded white) or untreated (shaded black) for 1h. Horizontal dotted lines represent RPKM expression threshold of 0.3. Horizontal bars represent mean value. (C) The number of read fragments mapping to non-neutrophil genes and (D) neutrophil-specific genes in each library. Data is shown as the average (±SEM) across three treatment conditions (UT, TNFα, GM-CSF) for each Donor and each isolation protocol. (E) The number of mapped reads in each dataset.
Fig 5.
Venn diagram showing differentially expressed genes between neutrophil samples prepared by either Polymorphprep™ (poly) or magnetic beads (bead).
Comparisons performed by Cufflinks using treatment specific paired-samples from two biological replicates. All genes displayed were significantly differentially expressed due to a higher RPKM in Polymorphprep prepared samples. Significance was calculated by Cuffdiff and adjusted for 5% false discovery rate by Benjamini-Hochberg correction for multiple-testing.
Fig 6.
Expression of chemokine and cytokine transcripts in neutrophils isolated by Polymorphprep (-P) or negative-selection by magnetic beads (-B).
(A) Cluster analysis showing expression levels (Log2 RPKM) in neutrophils incubated alone or in the presence of GM-CSF (GM) or TNF. (B-D) Expression levels of chemokine/cytokine genes with the highest level of expression in (B) untreated, (C) TNF and (D) GM-CSF treated neutrophils isolated by Polymorphprep (●) or negative selection (□).