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Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient.

Corkum CP, Ings DP, Burgess C, Karwowska S, Kroll W, Michalak TI - BMC Immunol. (2015)

Bottom Line: High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology.No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found.Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations.

View Article: PubMed Central - PubMed

Affiliation: Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, NL, A1B3V6, Canada. c.corkum@mun.ca.

ABSTRACT

Background: High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.

Results: The mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 10(6) cells/ml, 93.3%) were compatible to those obtained with Ficoll (1.34 × 10(6) cells/ml, 97.2%). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.

Conclusions: Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells' handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.

Show MeSH
Purity of immune cell subsets obtained from PBMC isolated by either Ficoll or CPT procedures. CD19+, CD8+, CD14+, and CD4+ cell subsets were sequentially separated from PBMC isolated by Ficoll and CPT protocols from the same donor and their purity was determined by flow cytometry using antibodies against surface markers specific for individual immune cell types. Filled histograms were given by appropriate immunoglobulin isotype controls. Gates for determining positivity were established using isotype controls so that ~99.0 % of events were negative
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Fig4: Purity of immune cell subsets obtained from PBMC isolated by either Ficoll or CPT procedures. CD19+, CD8+, CD14+, and CD4+ cell subsets were sequentially separated from PBMC isolated by Ficoll and CPT protocols from the same donor and their purity was determined by flow cytometry using antibodies against surface markers specific for individual immune cell types. Filled histograms were given by appropriate immunoglobulin isotype controls. Gates for determining positivity were established using isotype controls so that ~99.0 % of events were negative

Mentions: Accordingly, CD19+, CD8+, CD14+, and CD4+ cells were sequentially separated from each PBMC sample examined and their quantity and viability compared. Purities of CD19+, CD8+, CD14+, and CD4+ cells were evaluated by flow cytometry and were found to be usually 95 % or greater for cells derived from PBMC isolated by either Ficoll or CPT, as shown for cells from one of the donors in Fig. 4.Fig. 4


Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient.

Corkum CP, Ings DP, Burgess C, Karwowska S, Kroll W, Michalak TI - BMC Immunol. (2015)

Purity of immune cell subsets obtained from PBMC isolated by either Ficoll or CPT procedures. CD19+, CD8+, CD14+, and CD4+ cell subsets were sequentially separated from PBMC isolated by Ficoll and CPT protocols from the same donor and their purity was determined by flow cytometry using antibodies against surface markers specific for individual immune cell types. Filled histograms were given by appropriate immunoglobulin isotype controls. Gates for determining positivity were established using isotype controls so that ~99.0 % of events were negative
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4549105&req=5

Fig4: Purity of immune cell subsets obtained from PBMC isolated by either Ficoll or CPT procedures. CD19+, CD8+, CD14+, and CD4+ cell subsets were sequentially separated from PBMC isolated by Ficoll and CPT protocols from the same donor and their purity was determined by flow cytometry using antibodies against surface markers specific for individual immune cell types. Filled histograms were given by appropriate immunoglobulin isotype controls. Gates for determining positivity were established using isotype controls so that ~99.0 % of events were negative
Mentions: Accordingly, CD19+, CD8+, CD14+, and CD4+ cells were sequentially separated from each PBMC sample examined and their quantity and viability compared. Purities of CD19+, CD8+, CD14+, and CD4+ cells were evaluated by flow cytometry and were found to be usually 95 % or greater for cells derived from PBMC isolated by either Ficoll or CPT, as shown for cells from one of the donors in Fig. 4.Fig. 4

Bottom Line: High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology.No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found.Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations.

View Article: PubMed Central - PubMed

Affiliation: Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, NL, A1B3V6, Canada. c.corkum@mun.ca.

ABSTRACT

Background: High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.

Results: The mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 10(6) cells/ml, 93.3%) were compatible to those obtained with Ficoll (1.34 × 10(6) cells/ml, 97.2%). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.

Conclusions: Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells' handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.

Show MeSH