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Comparison between magnetic activated cell sorted monocytes and monocyte adherence techniques for in vitro generation of immature dendritic cells: an Egyptian trial.

El-Sahrigy SA, Mohamed NA, Talkhan HA, Rahman AM - Cent Eur J Immunol (2015)

Bottom Line: This has made them a major focus of interest in the conception of immunotherapeutic vaccine strategies.Cultured cells were stained with surface markers of iDCs: FITC-anti-CD14, PE-anti-CD11c, PE-anti-CD1a, PE-Cy5-anti-HLA-DR, and PE-anti-CD83 for flow cytometry analysis.Flow cytometry analysis revealed that the median interquartile percentages of MACS-DCs expressing CD14(-) was significantly higher compared to the DCs derived from monocytes separated by adherence (median 80.2 and interquartile range 77.7-80.7 vs. 40.2 and 30.4-40.6, respectively; p < 0.001).

View Article: PubMed Central - PubMed

Affiliation: Pediatrics Medical Research Division, National Research Centre, Egypt.

ABSTRACT

Introduction: Dendritic cells (DCs) are the most efficient antigen presenting cells, which are considered a central component of the immune system for their extraordinary capacity to initiate and modulate the immune responses elicited upon recognition of infectious agents. This has made them a major focus of interest in the conception of immunotherapeutic vaccine strategies.

Aim of the study: To standardise a protocol for in vitro differentiation of human peripheral blood monocytes into immature DCs (iDCs) upon treatment with specific growth factors and to compare two monocyte isolation methods including magnetic activated cell sorted (MACS) monocytes by CD14(+) immuno-magnetic beads and monocytes separated by adherence.

Material and methods: Immature DCs were generated from monocytes of human peripheral blood in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 after in vitro culture for seven days. Cultured cells were stained with surface markers of iDCs: FITC-anti-CD14, PE-anti-CD11c, PE-anti-CD1a, PE-Cy5-anti-HLA-DR, and PE-anti-CD83 for flow cytometry analysis.

Results: We found that the viability of MACS-DCs was higher than DCs derived from monocytes separated by adherence (median 50 and interquartile range 45-50 vs. 25 and 10-30, respectively; p < 0.001). Flow cytometry analysis revealed that the median interquartile percentages of MACS-DCs expressing CD14(-) was significantly higher compared to the DCs derived from monocytes separated by adherence (median 80.2 and interquartile range 77.7-80.7 vs. 40.2 and 30.4-40.6, respectively; p < 0.001). However, MACS-DCs expressed the same levels of CD11c, CD1a, and HLA-DR as well as CD83 compared to the DCs derived from monocytes separated by adherence with p value > 0.05.

Conclusions: Both positively selected monocytes and monocytes separated by adherence procedure gave the same results as regards cell surface marker expression, although the DCs purity and viability using MACS separated monocytes were better.

No MeSH data available.


Related in: MedlinePlus

Phenotype of DC generated after culture of MACS separated monocytes. Dead cells were excluded by gating on forward and side scatter properties, and the remaining viable cells were analysed. Representative dot plots of FCM and the gating strategy used: A) shows forward and side scatters to gate monocytes; B) shows CD14– cells were acquired after gating the monocytes population by forward and side scattered properties; C) gating approach for discrimination of CD14– CD11c+ HLA-DR+ populations; D) gating approach for discrimination of CD14– HLA-DR+ CD83– populations
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Figure 0003: Phenotype of DC generated after culture of MACS separated monocytes. Dead cells were excluded by gating on forward and side scatter properties, and the remaining viable cells were analysed. Representative dot plots of FCM and the gating strategy used: A) shows forward and side scatters to gate monocytes; B) shows CD14– cells were acquired after gating the monocytes population by forward and side scattered properties; C) gating approach for discrimination of CD14– CD11c+ HLA-DR+ populations; D) gating approach for discrimination of CD14– HLA-DR+ CD83– populations

Mentions: Figure 3 shows an example of the flow cytometry charts performed in our study for analysis of immature DCs phenotype after culture of MACS separated mono-cytes.


Comparison between magnetic activated cell sorted monocytes and monocyte adherence techniques for in vitro generation of immature dendritic cells: an Egyptian trial.

El-Sahrigy SA, Mohamed NA, Talkhan HA, Rahman AM - Cent Eur J Immunol (2015)

Phenotype of DC generated after culture of MACS separated monocytes. Dead cells were excluded by gating on forward and side scatter properties, and the remaining viable cells were analysed. Representative dot plots of FCM and the gating strategy used: A) shows forward and side scatters to gate monocytes; B) shows CD14– cells were acquired after gating the monocytes population by forward and side scattered properties; C) gating approach for discrimination of CD14– CD11c+ HLA-DR+ populations; D) gating approach for discrimination of CD14– HLA-DR+ CD83– populations
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472535&req=5

Figure 0003: Phenotype of DC generated after culture of MACS separated monocytes. Dead cells were excluded by gating on forward and side scatter properties, and the remaining viable cells were analysed. Representative dot plots of FCM and the gating strategy used: A) shows forward and side scatters to gate monocytes; B) shows CD14– cells were acquired after gating the monocytes population by forward and side scattered properties; C) gating approach for discrimination of CD14– CD11c+ HLA-DR+ populations; D) gating approach for discrimination of CD14– HLA-DR+ CD83– populations
Mentions: Figure 3 shows an example of the flow cytometry charts performed in our study for analysis of immature DCs phenotype after culture of MACS separated mono-cytes.

Bottom Line: This has made them a major focus of interest in the conception of immunotherapeutic vaccine strategies.Cultured cells were stained with surface markers of iDCs: FITC-anti-CD14, PE-anti-CD11c, PE-anti-CD1a, PE-Cy5-anti-HLA-DR, and PE-anti-CD83 for flow cytometry analysis.Flow cytometry analysis revealed that the median interquartile percentages of MACS-DCs expressing CD14(-) was significantly higher compared to the DCs derived from monocytes separated by adherence (median 80.2 and interquartile range 77.7-80.7 vs. 40.2 and 30.4-40.6, respectively; p < 0.001).

View Article: PubMed Central - PubMed

Affiliation: Pediatrics Medical Research Division, National Research Centre, Egypt.

ABSTRACT

Introduction: Dendritic cells (DCs) are the most efficient antigen presenting cells, which are considered a central component of the immune system for their extraordinary capacity to initiate and modulate the immune responses elicited upon recognition of infectious agents. This has made them a major focus of interest in the conception of immunotherapeutic vaccine strategies.

Aim of the study: To standardise a protocol for in vitro differentiation of human peripheral blood monocytes into immature DCs (iDCs) upon treatment with specific growth factors and to compare two monocyte isolation methods including magnetic activated cell sorted (MACS) monocytes by CD14(+) immuno-magnetic beads and monocytes separated by adherence.

Material and methods: Immature DCs were generated from monocytes of human peripheral blood in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 after in vitro culture for seven days. Cultured cells were stained with surface markers of iDCs: FITC-anti-CD14, PE-anti-CD11c, PE-anti-CD1a, PE-Cy5-anti-HLA-DR, and PE-anti-CD83 for flow cytometry analysis.

Results: We found that the viability of MACS-DCs was higher than DCs derived from monocytes separated by adherence (median 50 and interquartile range 45-50 vs. 25 and 10-30, respectively; p < 0.001). Flow cytometry analysis revealed that the median interquartile percentages of MACS-DCs expressing CD14(-) was significantly higher compared to the DCs derived from monocytes separated by adherence (median 80.2 and interquartile range 77.7-80.7 vs. 40.2 and 30.4-40.6, respectively; p < 0.001). However, MACS-DCs expressed the same levels of CD11c, CD1a, and HLA-DR as well as CD83 compared to the DCs derived from monocytes separated by adherence with p value > 0.05.

Conclusions: Both positively selected monocytes and monocytes separated by adherence procedure gave the same results as regards cell surface marker expression, although the DCs purity and viability using MACS separated monocytes were better.

No MeSH data available.


Related in: MedlinePlus