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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.

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Yield and viability of immune cell subsets prepared from PBMC isolated by either Ficoll or CPT protocol. a The mean number of CD19+, CD8+, CD14+, and CD4+ cells positively selected from Ficoll- and CPT-isolated PBMC collected from 6 healthy donors. The horizontal line denotes the means and each symbol represents an individual cell type isolated from Ficoll-PBMC (filled symbols) or CPT-PBMC (empty symbols) from each of 6 donors. b The mean viability of the same cell subsets. No significant differences (P < 0.05) in yields and viability of the immune cell subsets obtained from PBMC isolated by Ficoll and CPT protocols were found. Error bars indicate standard error of the mean
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Fig5: Yield and viability of immune cell subsets prepared from PBMC isolated by either Ficoll or CPT protocol. a The mean number of CD19+, CD8+, CD14+, and CD4+ cells positively selected from Ficoll- and CPT-isolated PBMC collected from 6 healthy donors. The horizontal line denotes the means and each symbol represents an individual cell type isolated from Ficoll-PBMC (filled symbols) or CPT-PBMC (empty symbols) from each of 6 donors. b The mean viability of the same cell subsets. No significant differences (P < 0.05) in yields and viability of the immune cell subsets obtained from PBMC isolated by Ficoll and CPT protocols were found. Error bars indicate standard error of the mean

Mentions: Also, no significant differences were found when comparing the number or yield of CD19+, CD8+, CD14+, and CD4+ cells from PBMC separated by Ficoll or CPT protocol (Fig. 5a). The mean numbers of positively selected B lymphocytes were 1.42 × 106 (SEM = 3.19 × 105) and 1.32 × 106 (SEM = 3.05 × 105) for Ficoll- and CPT-derived PBMC, respectively (P = 0.638). The mean number of CD8+ T cells obtained from Ficoll-isolated PBMC was 2.01 × 106 (SEM = 3.08 × 105), while that from CPT- isolated PBMC was 1.74 × 106 (SEM = 1.70 × 105) (P = 0.453). Comparable (P = 0.595) numbers of monocytes were also acquired from PBMC prepared by both techniques, with a mean yield of 3.87 × 106 (SEM = 6.01 × 105) and 3.63 × 106 (SEM = 5.28 × 105) cells from Ficoll- and CPT-derived PBMC, respectively. Lastly, the mean yield of CD4+ T lymphocytes was 2.48 × 106 (SEM = 4.39 × 105) for Ficoll-PBMC compared to 1.77 × 106 (SEM = 1.74 × 105) for CPT-PBMC with no significant difference detected (P = 0.165). These results indicated that the employment of the CPT protocol for PBMC collection does not alter the reciprocal proportions between isolated immune cell subsets or the surface expression of immune cell-defining surface molecules in comparison to the isolation of PBMC by the Ficoll method.Fig. 5


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)

Yield and viability of immune cell subsets prepared from PBMC isolated by either Ficoll or CPT protocol. a The mean number of CD19+, CD8+, CD14+, and CD4+ cells positively selected from Ficoll- and CPT-isolated PBMC collected from 6 healthy donors. The horizontal line denotes the means and each symbol represents an individual cell type isolated from Ficoll-PBMC (filled symbols) or CPT-PBMC (empty symbols) from each of 6 donors. b The mean viability of the same cell subsets. No significant differences (P < 0.05) in yields and viability of the immune cell subsets obtained from PBMC isolated by Ficoll and CPT protocols were found. Error bars indicate standard error of the mean
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Yield and viability of immune cell subsets prepared from PBMC isolated by either Ficoll or CPT protocol. a The mean number of CD19+, CD8+, CD14+, and CD4+ cells positively selected from Ficoll- and CPT-isolated PBMC collected from 6 healthy donors. The horizontal line denotes the means and each symbol represents an individual cell type isolated from Ficoll-PBMC (filled symbols) or CPT-PBMC (empty symbols) from each of 6 donors. b The mean viability of the same cell subsets. No significant differences (P < 0.05) in yields and viability of the immune cell subsets obtained from PBMC isolated by Ficoll and CPT protocols were found. Error bars indicate standard error of the mean
Mentions: Also, no significant differences were found when comparing the number or yield of CD19+, CD8+, CD14+, and CD4+ cells from PBMC separated by Ficoll or CPT protocol (Fig. 5a). The mean numbers of positively selected B lymphocytes were 1.42 × 106 (SEM = 3.19 × 105) and 1.32 × 106 (SEM = 3.05 × 105) for Ficoll- and CPT-derived PBMC, respectively (P = 0.638). The mean number of CD8+ T cells obtained from Ficoll-isolated PBMC was 2.01 × 106 (SEM = 3.08 × 105), while that from CPT- isolated PBMC was 1.74 × 106 (SEM = 1.70 × 105) (P = 0.453). Comparable (P = 0.595) numbers of monocytes were also acquired from PBMC prepared by both techniques, with a mean yield of 3.87 × 106 (SEM = 6.01 × 105) and 3.63 × 106 (SEM = 5.28 × 105) cells from Ficoll- and CPT-derived PBMC, respectively. Lastly, the mean yield of CD4+ T lymphocytes was 2.48 × 106 (SEM = 4.39 × 105) for Ficoll-PBMC compared to 1.77 × 106 (SEM = 1.74 × 105) for CPT-PBMC with no significant difference detected (P = 0.165). These results indicated that the employment of the CPT protocol for PBMC collection does not alter the reciprocal proportions between isolated immune cell subsets or the surface expression of immune cell-defining surface molecules in comparison to the isolation of PBMC by the Ficoll method.Fig. 5

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