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A novel method for isolation of human lung T cells from lung resection tissue reveals increased expression of GAPDH and CXCR6.

Day CE, Zhang SD, Riley J, Gant T, Wardlaw AJ, Guillen C - J. Immunol. Methods (2009)

Bottom Line: The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells.Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells.This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Lung Health, Department of Infection, Immunity and Inflammation, University of Leicester, Glenfield Hospital, Leicester, United Kingdom. caroline2day@gmail.com

ABSTRACT
Lung T lymphocytes are important in pulmonary immunity and inflammation. It has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5%+/-1.9% (n=11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8+/-2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells.

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A: Purity of lymphocytic cells (as determined by Kimura stain) at different stages of the purification process. The filtrate contained 28.64 ±2.66% lymphocytic cells, which increased to 53.2 ± 4.56% (p < 0.0001) after Percoll gradient and to 90.5 ± 1.91 (p = 0.0013, n = 11) after purification with microbeads to CD3. B: Representative FACS histograms showing the expression of CD3, from the top: filtrate, lymphocyte layer after Percoll and after MACS purification.
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fig1: A: Purity of lymphocytic cells (as determined by Kimura stain) at different stages of the purification process. The filtrate contained 28.64 ±2.66% lymphocytic cells, which increased to 53.2 ± 4.56% (p < 0.0001) after Percoll gradient and to 90.5 ± 1.91 (p = 0.0013, n = 11) after purification with microbeads to CD3. B: Representative FACS histograms showing the expression of CD3, from the top: filtrate, lymphocyte layer after Percoll and after MACS purification.

Mentions: Percoll is a modified silica gel that separates cells according to density and has been used to purify cell types from various tissues such as the liver and the gut (Svensson et al., 2002; Xu et al., 2002). Based on previous protocols (Svensson et al., 2002), we therefore used a discontinuous 40/70% Percoll gradient to separate macrophages from the lymphocytic cells in the lung filtrate. After centrifugation two layers were formed: A lower layer between the two Percoll concentrations which contained an enriched population of lymphocytes, whereas most alveolar macrophages and epithelial cells formed a layer on top of the gradient. The only cells which were pelleted at the bottom of the tube were erythrocytes. After Percoll density centrifugation, the purity of lymphocytic cells (by morphology) increased from 28.63 +/− 12.5% to 53.14 ± 21.5% (n = 22) (Fig. 1A). Flow cytometry analysis also showed a progressive increase in the percentage of CD3+ cells (Fig. 1B).


A novel method for isolation of human lung T cells from lung resection tissue reveals increased expression of GAPDH and CXCR6.

Day CE, Zhang SD, Riley J, Gant T, Wardlaw AJ, Guillen C - J. Immunol. Methods (2009)

A: Purity of lymphocytic cells (as determined by Kimura stain) at different stages of the purification process. The filtrate contained 28.64 ±2.66% lymphocytic cells, which increased to 53.2 ± 4.56% (p < 0.0001) after Percoll gradient and to 90.5 ± 1.91 (p = 0.0013, n = 11) after purification with microbeads to CD3. B: Representative FACS histograms showing the expression of CD3, from the top: filtrate, lymphocyte layer after Percoll and after MACS purification.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666793&req=5

fig1: A: Purity of lymphocytic cells (as determined by Kimura stain) at different stages of the purification process. The filtrate contained 28.64 ±2.66% lymphocytic cells, which increased to 53.2 ± 4.56% (p < 0.0001) after Percoll gradient and to 90.5 ± 1.91 (p = 0.0013, n = 11) after purification with microbeads to CD3. B: Representative FACS histograms showing the expression of CD3, from the top: filtrate, lymphocyte layer after Percoll and after MACS purification.
Mentions: Percoll is a modified silica gel that separates cells according to density and has been used to purify cell types from various tissues such as the liver and the gut (Svensson et al., 2002; Xu et al., 2002). Based on previous protocols (Svensson et al., 2002), we therefore used a discontinuous 40/70% Percoll gradient to separate macrophages from the lymphocytic cells in the lung filtrate. After centrifugation two layers were formed: A lower layer between the two Percoll concentrations which contained an enriched population of lymphocytes, whereas most alveolar macrophages and epithelial cells formed a layer on top of the gradient. The only cells which were pelleted at the bottom of the tube were erythrocytes. After Percoll density centrifugation, the purity of lymphocytic cells (by morphology) increased from 28.63 +/− 12.5% to 53.14 ± 21.5% (n = 22) (Fig. 1A). Flow cytometry analysis also showed a progressive increase in the percentage of CD3+ cells (Fig. 1B).

Bottom Line: The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells.Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells.This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Lung Health, Department of Infection, Immunity and Inflammation, University of Leicester, Glenfield Hospital, Leicester, United Kingdom. caroline2day@gmail.com

ABSTRACT
Lung T lymphocytes are important in pulmonary immunity and inflammation. It has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5%+/-1.9% (n=11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8+/-2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells.

Show MeSH
Related in: MedlinePlus