Jurkat Cells

Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3epsilon on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network.

Cell refractive index, an intrinsic optical parameter, is closely correlated with the intracellular mass and concentration. By combining optical phase-shifting interferometry (PSI) and atomic force microscope (AFM) imaging, we constructed a label free, non-invasive and quantitative refractive index of single cell measurement system, in which the accurate phase map of single cell was retrieved with PSI technique and the cell morphology with nanoscale resolution was achieved with AFM imaging. Based on the proposed AFM/PSI system, we achieved quantitative refractive index distributions of single red blood cell and Jurkat cell, respectively. Further, the quantitative change of refractive index distribution during Daunorubicin (DNR)-induced Jurkat cell apoptosis was presented, and then the content changes of intracellular biochemical components were achieved. Importantly, these results were consistent with Raman spectral analysis, indicating that the proposed PSI/AFM based refractive index system is likely to become a useful tool for intracellular biochemical components analysis measurement, and this will facilitate its application for revealing cell structure and pathological state from a new perspective.

The presence of relatively high levels of cellular protein contamination in density-purified virion preparations is a confounding factor in biochemical analyses of HIV and SIV produced from hematopoietic cells. A major source of this contamination is from vesicles, either microvesicles or exosomes, that have similar physical properties as virions. Thus, these particles can not be removed by size or density fractionation. Although virions and vesicles have similar cellular protein compositions, CD45 is excluded from HIV-1 yet is present in vesicles produced from hematopoietic cells. By exploiting this finding, we have developed a CD45 immunoaffinity depletion procedure that removes vesicles from HIV-1 preparations. While this approach has been successfully applied to virion preparations from several different cell types, some groups have concluded that "exosomes" from certain T cell lines, specifically Jurkat, do not contain CD45. If this interpretation is correct, then these vesicles could not be removed by CD45 immunoaffinity depletion. Here we show that dense vesicles produced by Jurkat and SupT1/CCR5 cells contain CD45 and are efficiently removed from preparations by CD45-immunoaffinity depletion. Also, contaminating cellular proteins were removed from virion preparations produced by these lines. Previously, the absence of CD45 from both "exosomes" and virions has been used to support the so called Trojan exosome hypothesis, namely that HIV-1 is simply an exosome containing viral material. The presence of CD45 on vesicles, including exosomes, and its absence on virions argues against a specialized budding pathway that is shared by both exosomes and HIV-1.

Cell viability, an essential measurement for cell therapy products, lacks traceability. One of the most common cell viability tests is trypan blue dye exclusion where blue-stained cells are counted via brightfield imaging. Typically, live and dead cells are classified based on their pixel intensities which may vary arbitrarily making it difficult to compare results. Herein, a traceable absorbance microscopy method to determine the intracellular uptake of trypan blue is demonstrated. The intensity pixels of the brightfield images are converted to absorbance images which are used to calculate moles of trypan blue per cell. Trypan blue cell viability measurements, where trypan blue content in each cell is quantified, enable traceable live-dead classifications. To implement the absorbance microscopy method, we developed an open-source AbsorbanceQ application that generates quantitative absorbance images. The validation of absorbance microscopy is demonstrated using neutral density filters. Results from four different microscopes demonstrate a mean absolute deviation of 3% from the expected optical density values. When assessing trypan blue-stained Jurkat cells, the difference in intracellular uptake of trypan blue in heat-shock-killed cells using two different microscopes is 3.8%. Cells killed with formaldehyde take up ~50% less trypan blue as compared to the heat-shock-killed cells, suggesting that the killing mechanism affects trypan blue uptake. In a test mixture of approximately 50% live and 50% dead cells, 53% of cells were identified as dead (±6% standard deviation). Finally, to mimic batches of low-viability cells that may be encountered during a cell manufacturing process, viability was assessed for cells that were 1) overgrown in the cell culture incubator for five days or 2) incubated in DPBS at room temperature for five days. Instead of making live-dead classifications using arbitrary intensity values, absorbance imaging yields traceable units of moles that can be compared, which is useful for assuring quality for biomanufacturing processes.