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Despite numerous separation methods of neutrophils from peripheral blood, isolation of sufficient quantities of high-purity viable cells for quantitative determination of neutrophil cytokines and their mRNA expression still remains an actual issue. The recommended multi-step purification methods significantly prolong the cell isolation process, potentially leading to cell activation or apoptosis and resulting in significant cell loss. Preliminary purification of neutrophils is the most critical stage in terms of time spent, and several additional manipulations with cells. To address this challenge, our study aimed to compare various methods of preliminary neutrophil isolation in order to select the optimal approach to obtaining a sufficient number of viable peripheral neutrophils.We studied the effects of three different protocols for preliminary isolation of cell suspensions: (a) centrifugation of whole blood at a single-step density gradient followed by sedimentation of red blood cells with dextran; (b) centrifugation of whole blood on a double density gradient; (c) rapid isolation of leukocytes using a reagent that promotes red blood cell aggregation. The cell counts and viability of purified neutrophils were tested at the final stage using negative immunomagnetic selection. Our study has shown that the methods used for preliminary neutrophil isolation significantly affect both the number and viability of the cells. The highest number of viable neutrophils was obtained using a conventional method of blood centrifugation at a density gradient followed by dextran sedimentation of red blood cells. However, the three studied methods of preliminary neutrophil isolation did not show statistically significant differences with respect to quantitative yield of viable cells after immunomagnetic isolation. These findings suggest that any of these methods may be applied, depending on capabilities and preferences of the researchers. In summary, our findings confirm previous studies indicating that the multistep process of neutrophil isolation allows for obtaining a high-purity cell suspension (> 99.1%) which can be used in future studies of their cytokine-secreting activity. However, such multi-stage isolation significantly reduces the yield of neutrophils, thus being critical for studying of initially small blood volumes.

Primary B- and T-lymphocytes and neoplastic cells of B- and T-cell origin are particularly hard to transfect with plasmids. Thus, functional and molecular studies in immunology and oncohematology require selection of cells after plasmid transduction, assuring obtaining homogeneous cell populations expressing plasmid-delivered molecules. The selection is usually achieved by incorporation of an antibiotic resistance gene or fluorescent protein into a plasmid and subsequent antibiotic selection or fluorescence-activated cell sorting (FACS). However, these cell sorting methods have significant drawbacks that limit their utilization. An ideal system should be fast, cheap and ensure efficient selection of transfected cells. In an attempt to deliver a sorting system meeting these requirements, we have generated a plasmid for retroviral gene delivery and expression, containing a recombinant human-murine CD4 gene, with a truncated intracellular domain to prevent undesired signaling events. After retroviral infection, cells expressing hmCD4 are selected by immunomagnetic anti-mCD4 beads. The system offers a very robust, efficient, fast and cost-saving cell separation solution. The vector is available for the academic community upon request.

Abstract Background The regional lymph node involvement is a major prognostic factor in patients with non-small cell lung cancer (NSCLC) undergoing surgical resection. Disease relapse is common, suggesting that early disseminated disease is already present in the regional lymph nodes at the time of surgery, and that the current nodal staging classification might be suboptimal. Early detection of disseminated tumor cells (DTCs) in lymph nodes could potentially enable identification of subcategories of patients with high risk of disease relapse. Method Lymph node samples were collected from 128 NSCLC patients at the time of surgery and the presence of DTCs determined by immunomagnetic selection (IMS) using the MOC31 antibody recognizing EpCAM. Results obtained with IMS were compared to the pathological staging obtained by histopathology. Associations between the presence of DTCs and clinicopathological variables and patient outcome were investigated. Results DTCs were detected in 40 % of the lymph node samples by IMS. Their presence was significantly associated with pN status as assessed by histopathology, and samples from 83 % of the patients with lymph node metastases (pN1-2) had detectable DTCs. In the group of patients who were negative for lymph node metastases by standard histopathology (pN0) DTCs were detected in 32 %. The presence of DTCs was not associated with any other clinicopathological variables. Patients with IMS-positive samples showed decreased relapse free survival compared to patients with IMS-negative samples, but the difference was not statistically significant. The pN status was significantly associated with both relapse free and overall survival, but the presence of DTCs had no prognostic impact in the subcategory of patients with pN0 status. Conclusion Our findings do not support further development of lymph node DTC detection for clinical use in early stage NSCLC.

Obtaining human tumor cell lines from fresh tumors is essential to advance our understanding of antitumor immune surveillance mechanisms and to develop new ex vivo strategies to generate an efficient anti-tumor response. The present study delineates a simple and rapid method for efficiently establishing primary cultures starting from tumor samples of different types, while maintaining the immuno-histochemical characteristics of the original tumor. We compared two different strategies to disaggregate tumor specimens. After short or long term in vitro expansion, cells analyzed for the presence of malignant cells demonstrated their neoplastic origin. Considering that tumor cells may be isolated in a closed system with high efficiency, we propose this methodology for the ex vivo expansion of tumor cells to be used to evaluate suitable new drugs or to generate tumor-specific cytotoxic T lymphocytes or vaccines.