Technique for single-step lymphocyte isolation from an endoscopic biopsy specimen for the diagnosis of gastrointestinal lymphoma.

In this paper, we introduce a simplified, one-step procedure for lymphocyte isolation from an endoscopically biopsied fragment. For lymphocyte isolation, an endoscopically harvested specimen and 5 mL of normal saline solution were placed in a wire mesh strainer set in a porcelain bowl. To obtain the lymphocyte suspension, the solid specimen was crushed using the rubber portion of a plunger of a 10 mL injection syringe. Flow cytometry was performed using the lymphocyte suspension. For validating our methods, the one-step lymphocyte isolation technique was used to perform flow cytometry on samples from 23 patients with (n = 12) or without (n = 11) gastrointestinal lymphoma. Flow cytometry of light chain expression was performed in all patient samples (feasibility: 100%). Sensitivity was 83.3% (10/12) and specificity was 100% (11/11). In conclusion, lymphocytes isolated from a single endoscopic biopsy specimen using our simplified and quick procedure are suitable for flow cytometry. Considering that flow cytometry has an important advantage of providing the results on the examination day itself, the results of this study suggest that flow cytometric analysis using our single-step lymphocyte isolation technique can be potentially used to diagnose lymphoma in the gastrointestinal mucosa. •We introduce a simplified, one-step procedure for lymphocyte isolation from an endoscopically biopsied fragment.•Our technique is feasible for flow cytometric analysis in patients with gastrointestinal lymphoma as well as those with gastrointestinal lesions that are suspected to be lymphoma.

Isolation and Characterization of Mouse Intrahepatic Lymphocytes by Flow Cytometry.

In the field of cellular immunology multicolor flow cytometry is a frequently applied method that allows for the simultaneously detection of multiple parameters on an individual cell basis. Flow cytometry can be used to characterize a wide range of immune cell subsets using fluorophore-conjugated antibodies to a wide range of cellular antigens. The isolation of immune cells from nonlymphoid tissue and their preparation for flow cytometry can be a challenging process with respect to immune cell yields and viability. Here we describe a method for the efficient isolation of viable mouse intrahepatic lymphocytes (IHL) from normal liver tissue and liver cancer and their subsequent characterization by multicolor flow cytometry.

An Optimized and Validated Method for Isolation and Characterization of Lymphocytes from HIV+ Human Gut Biopsies.

The gastrointestinal (GI) tract harbors most of the body's immune cells and is also a major HIV reservoir in ART-treated patients. To achieve a cure, most HIV-infected cells must be identified and eliminated. While obtaining gut biopsies is a relatively noninvasive method of sampling relevant tissue for monitoring HIV activity, immune cell isolation from these limited tissue samples has proven to be challenging. Enzymatic tissue digestion is required for maximal immune cell isolation from gut biopsies. However, these enzymatic digestions can also be detrimental for preservation of cellular surface markers that are required for accurate identification of various subsets of leukocytes. In this study, we describe an optimized protocol for isolation of lymphocytes from human gut biopsies. We also discuss our validation results, which show that compared with several other collagenase preparations, the use of CSLPA maintains high lymphocyte recovery while preserving the integrity of most cellular surface antigens that we tested. Importantly, chemokine receptors that are used to characterize various subsets of T cells, which are notorious for being digested during a typical enzymatic tissue digestion, are highly preserved using this protocol.

Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique.

Effective recovery of activated brain infiltrating lymphocytes is critical for investigations involving murine neurological disease models. To optimize lymphocyte recovery, we compared two isolation methods using brains harvested from seven-day Theiler's murine encephalomyelitis virus (TMEV) and TMEV-OVA infected mice. Brains were processed using either a manual dounce based approach or enzymatic digestion using type IV collagenase. The resulting cell suspensions from these two techniques were transferred to a percoll gradient, centrifuged, and lymphocytes were recovered. Flow cytometric analysis of CD45hi cells showed greater percentage of CD44hiCD62lo activated lymphocytes and CD19+ B cells using the dounce method. In addition, we achieved a 3-fold greater recovery of activated virus-specific CD8 T cells specific for the immunodominant Db:VP2121-130 and engineered Kb:OVA257-264 epitopes through manual dounce homogenization approach as compared to collagenase digest. A greater percentage of viable cells was also achieved through dounce homogenization. Therefore, we conclude that manual homogenization is a superior approach to isolate activated T cells from the mouse brain.

A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells.

The suppression assay is a commonly performed assay, measuring the ability of regulatory T cells (Treg) to suppress T cell proliferation. Most frequently, Treg are obtained from the peripheral blood or spleen. Lower yields are obtained by isolation from other tissues, rendering downstream suppression assays challenging to perform. Furthermore, the importance of suppressive subpopulations of Treg favours their isolation by fluorescent-activated cell sorting. Here we describe a method to isolate Treg from human tissues, using colorectal cancer tissue as an example. Treg suppressive capacity was further examined by expression of CCR5 to demonstrate the ability of our method to assess the suppressive capacity of regulatory T cell subsets. To optimise the standard suppression assay to achieve our research aims, the following modifications were made: Treg, isolated from tissues, were sorted directly into a well-plate.Responder T cells, which had been fluorescently-labelled prior to sorting, were added directly into the well-plate.Human Treg Suppression Inspector beads (Miltenyi Biotec Ltd, UK) provided a polyclonal stimulus for proliferation and were added to each well at a bead:lymphocyte ratio of 1:2. This method quantified the suppression of responder T cell proliferation by small numbers of strictly-defined Treg populations isolated from tissues.