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Updated: Aug 9, 2026
Isolation of Small Noncoding RNAs from Human Serum
Published on: June 19, 2014
A novel high-purity isolation method for human peripheral blood neutrophils permitting polymerase chain
C Lichtenberger1, S Zakeri, K Baier
1Clinic of Internal Medicine IV, Department of Gastroenterology and Hepatology, University of Vienna, Austria.
Abstract:
In the past few years, the role of polymorphonuclear neutrophils (PMN) in specific immune responses has gained significance due to their ability to express a variety of immunoregulatory molecules. However, controversial results concerning the potential of neutrophils for cytokine production have been obtained by sensitive molecular biological techniques. This problem might be related to contaminating leukocytes in conventionally isolated neutrophil suspensions as outlined by our study. We have established a novel method yielding highly purified neutrophils by combining a discontinuous Percoll gradient with fluorescence activated cell sorting of CD16bright cells. The latter step exploits the exceptionally high expression of Fc gammaRIIIB on PMN. Neutrophils could be enriched to homogeneity (> 99.9%) with a viability exceeding 90%. Contamination with NK cells or other lymphocytes, monocytes and eosinophils could be excluded as evaluated by reverse transcription-polymerase chain reaction (RT-PCR) with primers for HLA-DR, c-fms and CD52. The transcriptional potential of such purified neutrophils was confirmed by their ability to express MHC class II molecules after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Our method should permit studies of PMN at the mRNA level and future investigations concerning the specificity of immunoregulatory molecule synthesis by neutrophils.
Insights
Highly purified neutrophils (>99.9%) were obtained using a novel Percoll gradient and cell sorting method. This technique enables accurate studies of polymorphonuclear neutrophils (PMN) and their immunoregulatory molecule synthesis at the mRNA level.
Area of Science:
- Immunology
- Cell Biology
- Hematology
Background:
- Polymorphonuclear neutrophils (PMN) play a significant role in immune responses due to their immunoregulatory molecule expression.
- Controversial findings exist regarding neutrophil cytokine production, potentially due to leukocyte contamination in isolated samples.
- Accurate assessment of neutrophil function requires highly purified cell populations.
Purpose of the Study:
- To develop and validate a novel method for obtaining highly purified neutrophils.
- To overcome limitations of previous methods that may lead to inaccurate conclusions about neutrophil function.
- To enable reliable mRNA-level studies of neutrophil immunoregulatory molecule synthesis.
Main Methods:
- Combined discontinuous Percoll gradient with fluorescence-activated cell sorting (FACS) targeting CD16bright cells.
- Utilized the high expression of Fc gammaRIIIB on PMN for cell sorting.
- Assessed purity and contamination using RT-PCR for specific leukocyte markers (HLA-DR, c-fms, CD52).
Main Results:
- Achieved neutrophil homogeneity exceeding 99.9% with >90% viability.
- Effectively excluded contamination by NK cells, lymphocytes, monocytes, and eosinophils.
- Demonstrated transcriptional potential of purified neutrophils by MHC class II expression after GM-CSF stimulation.
Conclusions:
- The novel method provides highly purified neutrophils, crucial for accurate immunological studies.
- This technique resolves issues related to leukocyte contamination in neutrophil preparations.
- Enables future research into the mRNA expression and specific synthesis of immunoregulatory molecules by neutrophils.
