A method for prolonged imaging of motile lymphocytes

Abstract

With new imaging technologies and fluorescent probes, live imaging of cells in vitro has revolutionized many aspects of cell biology. A key goal now is to develop systems to optimize in vitro imaging, which do not compromise the physiological relevance of the study. We have developed a methodology that contains non-adherent cells within the field of view. 'Cell paddocks' are created by generating an array of microgrids using polydimethylsiloxane. Each microgrid is up to 250Â250 lm 2 with a height of 60 lm. Overlayed cells settle into the grids and the walls restrict their lateral movement, but a contiguous supply of medium between neighboring microgrids facilitates the exchange of cytokines and growth factors. This allows culture over at least 6 days with no impact upon viability and proliferation. Adaptations of the microgrids have enabled imaging and tracking of lymphocyte division through multiple generations of long-term interactions between T lymphocytes and dendritic cells, and of thymocytestromal cell interactions. Live imaging has enabled huge progress in understanding the molecular activities underlying cellular processes, most notably activities such as migration 1 and cell-cell communication. 2 Many of our advances have related to cells such as fibroblasts, neuronal and epithelial cells, whose relatively large size, slow movement and tendency to arrange as monolayers make them amenable to longterm imaging. Lymphocytes, however, provide challenges for live imaging that have restricted studies in some aspects of lymphocyte biology to date. In particular, lymphocytes (mature T and B cells and thymocytes or T-cell precursors) migrate rapidly (up to 25 mm min À1 ) (Beltman et al. 3 ) and are likely to leave the field of view during highresolution imaging over more than a few minutes. In addition, T cells tend to undergo homotypic interactions in vitro, forming large clumps that complicate image analysis. 4 Diluting the cells (for instance, single cell cultures in Terasaki wells) is used to overcome clumping and facilitate tracking of individual cells, but does not facilitate highresolution imaging, and is often problematic because of the dependence of many cells on autocrine signals that cannot be maintained at low dilutions.T cells are activated by the presentation of antigen by professional antigen-presenting cells. This process involves morphological rearrangements including the formation of a structure at the interface between the two cells referred to as the immunological synapse. 5 In vivo imaging of T-cell activation has revealed that T cells can remain in contact with antigen-presenting cells for hours, 6 and can still interact at the point of cell division more than 20 h after activation. 7,8 With recent observations that T cells can undergo asymmetric cell division to dictate cell fate, 9 there is strong incentive to develop new approaches to in vitro imaging of T-cell interactions with dendritic cells over hours and even days. Many studies observing antigen presentation have overcome the proble...

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

“…Images were captured at 5 min intervals over a 2-h period using a  60 lens and 2  2 binning of the camera. Non-adherent CLs were cultured in polydimethylsiloxane microgrids 52 (125  125 mm by 60 mm deep and containing 32 chambers) positioned in 8-well microslides (Ibidi, Munich, Germany). The microgrids have walls but no lid and act as corrals, physically confining a small number of cells in each chamber.…”

Regulation of asymmetric cell division and polarity by Scribble is not required for humoral immunity

“…Naive B cells were cultured in LPS for 24 h, and before cell division had occurred ( Supplementary Fig. S4), single B cells were transferred into 125 mm  125 mm cell paddocks 26 in the presence of LPS and PE-conjugated anti-CD138 antibody. Using environmentcontrolled time-lapse microscopy, single cells were then imaged for 4 days.…”

“…Finally, Scribble has been implicated in lymphocyte migration and we reasoned that the ability of Scribble null/null B cells to migrate and interact with each other might contribute to B lymphocyte function or ability to interact with signals from neighbouring cells. B cells were isolated from Scribble null/null mice, cultured with LPS in 8-well chamber slides lined with micro-paddocks 26 and migration tracked by live cell microscopy for 2 days. Contrary to previous findings, Scribble null/null B cells displayed no difference in migration in vitro as determined by single-cell tracking (Figs 3e,f).…”

Converse Smith-Martin cell cycle kinetics by transformed B lymphocytes

“…Single clone lines were established that demonstrated stable expression of each FUCCI component up to and exceeding 30 days (Figure 1(a) and S1). Having established FUCCI-J558 and FUCCI-I.29 lines we adapted a single cell imaging system previously used to investigate cell cycle lengths [17,25,26]. Single cells seeded in microgrids were filmed over 60 hours to observe 1–3 division rounds, and we developed an imaging analysis pipeline (described in Methods) to measure the onset of G1 (t redmax ) and S/G2/M (t div -t redmax ) (Figure 1(b)).…”