Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.
Part 1: Transfection of neurons using Lipofectamine 2000 (Invitrogen)
Equipment set-up:
Rat or mouse hippocampal neurons are cultured according to Kaech and Banker, 2006 [4]. The typical cell density used for transfections is 250,000 cells/6-cm dish. Required reagents and equipment include 50 mM kynurenic acid, regular benchtop tube holder, a benchtop cooler (best to keep Lipofectamine reagent cold),
Lipofectamine reagent, MEM, microfuge tubes, micropipetters, and sterile forceps.
Procedure:
Part 2: Live imaging of GFP-tagged dense-core vesicles in cultured hippocampal neurons
Equipment set-up:
Procedure:
Part 3: Representative Results:
Live cell imaging observations typically consist of videos (also known as “stacks” of images) that show movement of fluorescently-tagged organelles within the field of view (Figure 1A). Accompanying each video are often supporting images that illustrate orientation of the field of view with respect to the cell body, level of protein expression and/or the health of the cell. Videos of transport can be subjected to quantitative analysis by transformation to kymographs (Figure 1B). Kymographs are images that illustrate particle movement by juxtaposing line scans where each time point is represented by a column in the kymograph. Figure 2 demonstrates how two particles moving in opposite direction are represented in a kymograph. Further analysis of kymographs can provide information about particle flux, velocity, run length, reversals, and stationary particles.
Figure 1. Representative Live Cell Imaging Observations. (A) Selected frames from a video of mCherry- tagged Tissue Plasminogen Activator (TPA) transport. Individual particles moving in the anterograde (green arrows) and retrograde (red arrows) directions are indicated. (B) Kymographs illustrating TPA-mCherry movement in the axon. Horizontal lines in kymographs represent stationary objects, while diagonal lines represent organelles moving away from (positive slope) or toward (negative slope) the cell body. The long green and red arrows show the runs corresponding to the particles in (A). Changes in transport due to the microtubule depolymerizing reagent, nocodazole, is also illustrated with a kymograph. Note the reduction in moving organelles by the absence of diagonal lines.
Live cell imaging is a challenging, but powerful technique for the direct observation of organelle transport in cultured neurons. Difficulties can arise upstream of the procedure with poor neuron health due to culture complications. Therefore, cell health (for examples see ref. 4) should be assessed before and after transfection by observing the coverslips while in growth medium using a tissue culture light microscope. The process of transferring neuron-containing coverslips to the imaging chamber can be stressful to the cells, thus it is important to exercise care when manipulating the coverslips. It can be common for healthy-looking cells to show no transport due to delays in the procedure, or incomplete pre-heating of equipment or equilibration of the imaging media. For transport analysis the axonal and dendritic orientation must be known, i.e. distinguishing anterograde and retrograde transport. It is therefore critical to be sure that the location of a cell body is determined and that a continuous neuronal segment can be seen connecting the region of interest to the cell body. Often saving overlapping images of soluble GFP, or informatively naming the saved video file, is sufficient to ascertain the processes’ orientation for analyses, e.g., “Cell1CellBodyUpperLeft”.
We thank Harald Hutter and Helena Decker for their careful reading of this manuscript. We also thank Reg Sidhu from Leica Microsystems for his technical expertise. This research was supported by the National Sciences and Engineering Council of Canada, Award #327100-06, and the Simon Fraser University Faculty of Science.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
MEM-Eagle with Earle salts and L-glutamine | Reagent | Mediatech | 10-010-CV | |
Lipofectamine 2000 | Reagent | Invitrogen | 11668-027 | Transfection reagent |
10X Hanks with Ca2+ and Mg2+ | Reagent | Gibco/Invitrogen | 14185-052 | Live-imaging medium |
1M HEPES | Reagent | Gibco/Invitrogen | 15630-130 | Live-imaging medium |