Limits...
Whose Gene Is It Anyway? The Effect of Preparation Purity on Neutrophil Transcriptome Studies.

Thomas HB, Moots RJ, Edwards SW, Wright HL - PLoS ONE (2015)

Bottom Line: The level of contamination was assessed by morphology and flow cytometry.RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα).The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types.

View Article: PubMed Central - PubMed

Affiliation: Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

ABSTRACT
Protocols for the isolation of neutrophils from whole blood often result in neutrophil preparations containing low numbers (~5%) of contaminating leukocytes, and it is possible that these contaminating cells contribute to highly sensitive assays that measure neutrophil gene expression (e.g. qPCR). We investigated the contribution of contaminating leukocytes on the transcriptome profile of human neutrophils following stimulation with inflammatory cytokines (GM-CSF, TNFα), using RNA-Seq. Neutrophils were isolated using Polymorphprep or the StemCell untouched neutrophil isolation kit (negative selection of "highly pure" neutrophils). The level of contamination was assessed by morphology and flow cytometry. The major source of contamination in Polymorphprep neutrophil preparations was from eosinophils and was highly donor dependent. Contaminating cells were largely, but not completely, absent in neutrophil suspensions prepared using negative selection, but the overall yield of neutrophils was decreased by around 50%. RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα). The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types. This work demonstrates that low numbers (<5%) of contaminating leukocytes in neutrophil preparations contribute very little to the overall gene expression profile of cytokine-stimulated neutrophils, and that protocols for the isolation of highly pure neutrophils result in significantly lower yields of cells which may hinder investigations where large numbers of cells are required or where volumes of blood are limited.

No MeSH data available.


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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4581699&req=5

pone.0138982.g001: 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.

Mentions: The levels of contamination of neutrophil preparations following Polymorphprep isolation or negative selection from the blood of Donors 1 and 2 were quantified by cytospin and flow cytometry. Cytospins were prepared and the mean number of neutrophils, monocytes, lymphocytes and eosinophils quantified in four separate fields of view (x20 magnification). A minimum of 100 cells were counted in each field (Fig 1A and 1B). Similar levels of lymphocyte and monocyte contamination were seen in both donors (Fig 1A and 1B). However, the greatest differences between donors was the number of eosinophils following Polymorphprep isolation, with Donor 1 having 1% eosinophils and Donor 2 having 15% eosinophils (Fig 1A and 1B). The overall purity of neutrophils was higher following negative selection than with Polymorphprep (Donor 1: 96% Polymorphprep, 97.5% negative selection; Donor 2: 83% Polymorphprep, 99% negative selection). Morphological analysis of levels of apoptosis immediately after isolation revealed that neither isolation method resulted in significant levels of apoptotic neutrophils (<1%, data not shown).


Whose Gene Is It Anyway? The Effect of Preparation Purity on Neutrophil Transcriptome Studies.

Thomas HB, Moots RJ, Edwards SW, Wright HL - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581699&req=5

pone.0138982.g001: 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.
Mentions: The levels of contamination of neutrophil preparations following Polymorphprep isolation or negative selection from the blood of Donors 1 and 2 were quantified by cytospin and flow cytometry. Cytospins were prepared and the mean number of neutrophils, monocytes, lymphocytes and eosinophils quantified in four separate fields of view (x20 magnification). A minimum of 100 cells were counted in each field (Fig 1A and 1B). Similar levels of lymphocyte and monocyte contamination were seen in both donors (Fig 1A and 1B). However, the greatest differences between donors was the number of eosinophils following Polymorphprep isolation, with Donor 1 having 1% eosinophils and Donor 2 having 15% eosinophils (Fig 1A and 1B). The overall purity of neutrophils was higher following negative selection than with Polymorphprep (Donor 1: 96% Polymorphprep, 97.5% negative selection; Donor 2: 83% Polymorphprep, 99% negative selection). Morphological analysis of levels of apoptosis immediately after isolation revealed that neither isolation method resulted in significant levels of apoptotic neutrophils (<1%, data not shown).

Bottom Line: The level of contamination was assessed by morphology and flow cytometry.RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα).The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types.

View Article: PubMed Central - PubMed

Affiliation: Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

ABSTRACT
Protocols for the isolation of neutrophils from whole blood often result in neutrophil preparations containing low numbers (~5%) of contaminating leukocytes, and it is possible that these contaminating cells contribute to highly sensitive assays that measure neutrophil gene expression (e.g. qPCR). We investigated the contribution of contaminating leukocytes on the transcriptome profile of human neutrophils following stimulation with inflammatory cytokines (GM-CSF, TNFα), using RNA-Seq. Neutrophils were isolated using Polymorphprep or the StemCell untouched neutrophil isolation kit (negative selection of "highly pure" neutrophils). The level of contamination was assessed by morphology and flow cytometry. The major source of contamination in Polymorphprep neutrophil preparations was from eosinophils and was highly donor dependent. Contaminating cells were largely, but not completely, absent in neutrophil suspensions prepared using negative selection, but the overall yield of neutrophils was decreased by around 50%. RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα). The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types. This work demonstrates that low numbers (<5%) of contaminating leukocytes in neutrophil preparations contribute very little to the overall gene expression profile of cytokine-stimulated neutrophils, and that protocols for the isolation of highly pure neutrophils result in significantly lower yields of cells which may hinder investigations where large numbers of cells are required or where volumes of blood are limited.

No MeSH data available.