Flow Cytometry (FACS) Application Experiments

Flow cytometry is a general method for analyzing microparticles such as cells, bacteria and other microorganisms with high efficiency, integrating optics, fluidics, electronics, and computer technology. With the application of all kinds of numerous advanced technologies, flow cytometers are getting becoming smaller, cheaper, faster, more integrated and better in performance.

In clinical laboratories, it has been used for some applications as follows: analysis of leukaemias and lymphomas, detection of minimal residual disease, stem cell enumeration, T cell cross-match, postoperative monitoring, detection of autoantibodies, HIV infection, foeto-maternal haemorrhage, immunodeficiency diseases, paroxysmal nocturnal haemoglobinuria, reticulocyte analysis, contaminating leucocytes, measurements on red blood cells (RBCs), platelet counting and function, quality control, and so on.

In immunology, it can be used for analyzation of lymphocyte sub-populations, detection of cytotoxic T lymphocyte (CTL), detection of cytokines in cell and body fluid, observation of infection and its therapeutic effect, and so on.

In cell biology, flow cytometry also can be used for detection of cell apoptosis, cell cycles, the concentration of Ca2+, incellular pH, cell sorting, and so on.

Detection of RANKL produced by synovial B cells of RA patients

Receptor activator of nuclear factor kappa-B ligand (RANKL) is important in bone metabolism, which is the key cytokine driving bone destruction. The expression of RANKL can be detected by flow cytometry. Peripheral blood and synovial fluid mononuclear cells were co-labelled with anti-CD19 and either isotype matched control or anti-RANKL antibody. The results showed that only a very small proportion of B cells expressed RANKL in the peripheral blood of rheumatoid arthritis (RA) patients, a significantly higher proportion of B cells from synovial fluid samples expressed RANKL on their surface. This is the first study to report that B cells contribute to RANKL production in the inflamed rheumatoid joint, which may suggest that depletion of RANKL-expressing B cells may contribute to the inhibition of bone erosion by rituximab.

Reference: Lorraine Yeo, Kai-Michael Toellner, Mike Salmon, Andrew Filer, Christopher D Buckley, Karim Raza, Dagmar Scheel-Toellner. Cytokine mRNA profi ling identifi es B cells as a major source of RANKL in rheumatoid arthritis. British Medical Journal 2011, doi:10.1136/ard.2011.153312

Cell surface receptors detection in osteosarcoma

Osteosarcoma is the eighth most common form of childhood cancer, which has a numerous cell signaling receptors. The current study has sought to characterize and quantify the expression of cell surface receptors across a variety of osteosarcoma cell lines using flow cytometry.

The receptors tested includes human epidermal growth factor receptor (HER)-2, HER-3, HER-4, insulin-like growth factor 1 receptor (IGF-1R), IGF-2R, insulin receptor (IR), vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, VEGFR-3, c-Met, fibroblast growth factor receptor (FGFR)-2, FGFR-3, and platelet-derived growth factor receptor (PDGFR)-β.

The results showed that IGF-2R has significant overexpression. A variable expression pattern was seen for c-Met, PDGFR-β, IR, IGFR-1, HER-2, and VEGFR-3, suggesting that these receptors may contribute to osteosarcoma aggressiveness and biological heterogeneity and may serve as potential targets within a subset of tumors. Associated receptor expression may provide new insight into common regulatory factors or pathways. Targeting either common factors or targeting multiple specific receptors may have therapeutic relevance.

Reference: Hassan SE, Bekarev M, Kim MY, Lin J, Piperdi S, Gorlick R, Geller DS. Cell surface receptor expression patterns in osteosarcoma. Cancer 2011, doi: 10.1002/cncr.26339.

Detection methods of the protein CD44 in breast cancer cell

CD44 is a cell membrane receptor that links hyaluronate to the cytoskeleton ankyrin to mediate signal transduction. CD44 also plays a role in cell migration, differentiation, and survival signaling, which is important both to normal cells and cancer cells. Blocking of CD44 decreases the malignant activities of the tumor.

The expression of CD44 protein in human breast cancer cell line MCF-7/CD44st cells (MCF-7 cells transfected with pcDNA3.1-CD44st), MCF-7 cells, and MCF-7/neo cells (MCF-7 cells transfected with pcDNA3.1) were detected using flow cytometry analysis. Among these cell lines, only MCF-7/CD44st cells expressed a high level CD44 protein and the cells transfected stablely with pcDNA3.1-CD44st were constructed successfully.

This detection method contribute to demonstrate that HA could interact with the new CD44st and regulate the expression of MMP-2 and MMP-9, which could increase the invasion capability of MCF-7 cells through the Ras/MAPK signaling pathway.

Reference: Fang XJ, Jiang H, Zhao XP, Jiang WM. The role of a new CD44st in increasing the invasion capability of the human breast cancer cell line MCF-7. BMC Cancer 2011, 11(1):290.

HLA class I down-regulate on peripheral T lymphocytes and NK cells of gastrointestinal tumor

Human leukocyte antigens (HLA) class I are cell surface glycoproteins which play important roles in the regulation of immune responses. It is known that total or partial loss of HLA class I on tumor cells is one of the main mechanisms of tumor escape. T lymphocytes and NK cells, as the two critical effector cells of immune defense, also express plentiful HLA class I.

The percentage of T lymphocytes and NK cells and levels of HLA class I on their surface can be determined by flow cytometry. The result showed that percentages of T lymphocytes and NK cells in peripheral-blood mononuclear cells did not vary with age. Meanwhile, the expression level of HLA class I on peripheral T lymphocytes and NK cells was not affected by age or gender, either. But compared with the low-risk group, there was a significant reduction of HLA class I on peripheral T lymphocytes and NK cells in the high-risk group.

HLA class I on peripheral T lymphocytes and NK cells may be involved in tumorigenesis and development of gastrointestinal tumor, and understanding their changes in expression may provide new insights into the mechanism of tumor immunity.

Figure 1 The gates of peripheral-blood T lymphocytes, NK cells and HLA class I expression. A: Total lymphocytes were separated from peripheral-blood (Gate 1). B: T lymphocytes (Gate 2) and NK cells (Gate 3) were separated from total lymphocytes. C: Mean fluorescence intensity of HLA class I on T lymphocytes. D: Mean fluorescence intensity of HLA class I on NK cells.

Reference: Zhang ZM, Li YJ, Guan X, Yang XY, Gao XM, Yang XJ, Wang LS, Zou X. Down-regulation of human leukocyte antigens class I on peripheral T lymphocytes and NK cells from subjects in region of high-incidence gastrointestinal tumor. Chin Med J (Engl) 2011, 124(12):1813-7.

Cell Sorting of Pneumocystis carini

Pneumocystis jirovecii provokes a severe pneumonia (PcP), which continues to be of great concern inimmunocompromised HIV-negative individuals such as patients with primary immunodeficiency, patients receiving immunosup-pressive therapies for malignancies, organ transplantations orautoimmune diseases. Using the fungal species infecting the rat, and sorting its life cycle stages by flow cytometry in order to study their transmission efficiency, their transcriptome or their ploidy level, is a step way further to understanding its life cycle.

The result showed that most of P.carinii organisms were haploid. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores and each spore evolves into a trophic form. The ploidy data of P. carinii lifecycle stages shed new light on the complexity of its modes of proliferation.

Figure 1 Representative cell cycle analysis histograms of sorted P. carinii organisms. DNA contents of (A) all sorted P. carinii life cyclestages; (B) sorted P. carinii trophic forms; (C) haploid S. cerevisiae reference strain; (D) sorted P. carinii cystic forms; (E) diploid S. cerevisiae referencestrain. Figure 2 Interpretation scheme of P. carinii life cycle and cell cycle.

Reference: Anna Martinez, El Moukhtar Aliouat, Annie Standaert-Vitse, et al. Ploidy of Cell-Sorted Trophic and Cystic Forms of Pneumocystis carinii. PLoS One. 2011;6(6):e20935.

Tea components effect on apoptosis and cell cycle of gastric cancer cell

Tea is popular widespread because of its possible beneficial health effects. Generally, tea can be classified according to the production method as unfermented tea (green tea), fully fermented tea (black tea) and post-fermented tea (pu-erh tea). The chemical components in these three kinds of tea extracts were different. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. This differentiation has distinct effect on apoptosis and cell cycle arrest.

The results showed that catechins, which is the main tea phenolic compounds, could induce early apoptosis of gastric cancer cell lines SGC-7901 higher, and during the fermentation process, the content of phenolic compounds reduced. Cell cycle results showed that the proportion of G2/M phase cells decreased with the fermentation process, which may be due to the oxidation of tea polyphenols, and increase of theabrownins. In addition, the results also showed that tea extract and their constituents have lower cytotoxicity to normal CCC-HEL-1 cells. Cell cycle was not affected with the high concentration treatment. These results suggest that tea extract and their constituents could be a candidate agent for the therapy of gastric cancer.

Figure 1 Flow cytometric analysis of cell apoptosis induced by treatment for 48 h in SGC-7901 and CCC-HEL-1 cells. Figure 2 Cell cycle analysis of SGC-7901 and CCC-HEL-1 cells after treatment with three kinds of tea extract and their main constituents.

Reference: Hang Zhao, Min Zhang, Lu Zhao, Ya-kun Ge, Jun Sheng and Wei Shi. Changes of Constituents and Activity to Apoptosis and Cell Cycle During Fermentation of Tea. Int. J. Mol. Sci. 2011 12: 1862-1875; doi:10.3390/ijms12031862