Journal of Biological Methods (JBM)
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Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human T-cells
No full textChimeric antigen receptors (CARs) are genetically delivered fusion molecules that elicit T-cell activation upon binding of a native cell surface molecule. These molecules can be used to generate a large number of memory and effector T-cells that are capable of recognizing and attacking tumor cells. Most commonly, stable CAR expression is achieved in T-cells using retroviral vectors. In the method described here, retroviral vectors are packaged in a two-step procedure. First, H29D human retroviral packaging cells (a derivative of 293 cells) are transfected with the vector of interest, which is packaged transiently in vesicular stomatitis virus (VSV) G pseudotyped particles. These particles are used to deliver the vector to PG13 cells, which achieve stable packaging of gibbon ape leukaemia virus (GALV)-pseudotyped particles that are suitable for infection of human T-cells. The key advantage of the method reported here is that it robustly generates polyclonal PG13 cells that are 100% positive for the vector of interest. This means that efficient gene transfer may be repeatedly achieved without the need to clone individual PG13 cells for experimental pre-clinical testing. To achieve T-cell transduction, cells must first be activated using a non-specific mitogen. Phytohemagglutinin (PHA) provides an economic and robust stimulus to achieve this. After 48-72 h, activated T-cells and virus-conditioned medium are mixed in RetroNectin-coated plasticware, which enhances transduction efficiency. Transduced cells are analyzed for gene transfer efficiency by flow cytometry 48 h following transduction and may then be tested in several assays to evaluate CAR function, including target-dependent cytotoxicity, cytokine production and proliferation
Method for the quantitative measurement of collecting lymphatic vessel contraction in mice
No full textCollecting lymphatic vessels are critical for the transport of lymph and its cellular contents to lymph nodes for both immune surveillance and the maintenance of tissue-fluid balance. Collecting lymphatic vessels drive lymph flow by autonomous contraction of smooth muscle cells that cover these vessels. Here we describe methods using intravital microscopy to image and quantify collecting lymphatic vessel contraction in mice. Our methods allow for the measurement of the strength of lymphatic contraction of an individual lymphangion in a mouse, which has not yet been demonstrated using other published methods. The ability to study murine collecting lymphatic vessel contraction—using the methods described here or other recently published techniques—allows the field to dissect the molecular mechanisms controlling lymphatic pumping under normal and pathological conditions using the wide variety of molecular tools and genetic models available in the mouse. We have used our methods to study lymphatic contraction in physiological and inflammatory conditions. The methods described here will facilitate the further study of lymphatic function in other pathological conditions that feature lymphatic complications
Isolating astrocytes and neurons sequentially from postnatal murine brains with a magnetic cell separation technique
No full textUnderstanding the mechanism of developmental brain injury is crucial for the progress of discovering neuroprotective strategies and interventions. However, the pathophysiology is complex which involves interactions and crosstalk of diverse neural cell types. Isolating viable and pure populations of these brain cells is a valuable tool to study the particular cell properties and understand the physiologic and pathophysiologic mechanisms. Here we present a magnetic cell sorting approach to separate astrocytes and neurons sequentially from the same neonatal (postnatal day 9 or 10) CD-1 mouse brain samples. The procedure which involves positive selection of astrocytes by the ACSA-2 antibody followed by a negative depletion of non-neuronal cells from the flow through yields relatively enriched neuronal cells. The sorted fractions are highly pure and viable and can be used for further applications and analyses