An Evaluation of T-Cell Functionality After Flow Cytometry Sorting Revealed p38 MAPK Activation.

Cell alterations during isolation and preparation for flow cytometry cell sorting by antibodies, temperature, homogenization, buffer composition and mitogens are well known. In contrast, little is known about cell alteration caused by the instrument or the sorting process itself. We systematically evaluated cellular responses to different sorter-induced physical forces. In summary, flow cytometry cell-sorting induced forces can affect cellular signaling cascades, especially the MAPK p38. Functional assays, related to the p38 MAPK pathway, of human primary T cells after flow cytometry sorting did lead to minor physiological modulation but no functional impairments. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Efficient immunoaffinity chromatography of lymphocytes directly from whole blood.

We show that defined lymphocytes can be rapidly purified by immunoaffinity chromatography starting directly from whole blood. The method relies on low-affinity Fab-fragments attached to a column-matrix combined with the reversible Strep-tag technology. Compared to established cell enrichment protocols, the Strep-tag affinity chromatography of cells is independent of erythrocyte lysis or centrifugation steps, allowing for simple cell-enrichment with good yields, high purities, and excellent functionality of purified cells.

Sensing dynamic cytoplasm refractive index changes of adherent cells with quantitative phase microscopy using incorporated microspheres as optical probes.

The intracellular refractive index is an important parameter that describes the optical density of the cytoplasm and the concentration of the intracellular solutes. The refractive index of adherently grown cells is difficult to access. We present a method in which silica microspheres in living cells are used to determine the cytoplasm refractive index with quantitative phase microscopy. The reliability of our approach for refractive index retrieval is shown by data from a comparative study on osmotically stimulated adherent and suspended human pancreatic tumor cells. Results from adherent human fibro sarcoma cells demonstrate the capability of the method for sensing of dynamic refractive index changes and its usage with microfluidics.

Contrast-enhanced digital holographic imaging of cellular structures by manipulating the intracellular refractive index.

The understanding of biological reactions and evaluation of the significance for living cells strongly depends on the ability to visualize and quantify these processes. Digital holographic microscopy (DHM) enables quantitative phase contrast imaging for high resolution and minimal invasive live cell analysis without the need of labeling or complex sample preparation. However, due to the rather homogeneous intracellular refractive index, the phase contrast of subcellular structures is limited and often low. We analyze the impact of the specific manipulation of the intracellular refractive index by microinjection on the DHM phase contrast. Glycerol is chosen as osmolyte, which combines high solubility in aqueous solutions and biological compatibility. We show that the intracellular injection of glycerol causes a contrast enhancement that can be explained by a decrease of the cytosolic refractive index due to a water influx. The underlying principle is proven by experiments inducing cell shrinkage and with fixated cells. The integrity of the cell membrane is considered as a prerequisite and allows a reversible cell swelling and shrinking within a certain limit. The presented approach to control the intracellular phase contrast demonstrated for the example of DHM opens prospects for applications with other quantitative phase contrast imaging methods.