Microglial cells have important roles in maintaining brain homeostasis, and they are implicated in multiple brain diseases. There is currently interest in investigating microglial migration that results in cell accumulation at focal sites of injury. Here we describe a protocol for rapidly triggering and monitoring microglial migration by using a micropipette assay. This protocol is an adaptation of the axon turning assay using microglial cells. Chemoattractants released from the micropipette tip produce a chemotactic gradient that induces robust microglial migration. In combination with microscopic imaging, this assay allows simultaneous recording of cell movement and subcellular compartment trafficking, along with quantitative analysis. The actual handling time for the assay takes ∼2–3 h in total. The protocol is simple, inexpensive and convenient to set up, and it can be adopted to examine cell migration in multiple cell types, including cancer cells with a wide range of chemical signals.
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Systemic pro-inflammatory response facilitates the development of cerebral edema during short hypoxia
Journal of Neuroinflammation Open Access 11 March 2016
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This work was supported by grants from the Major State Basic Research Program of China (no. 2011CB504400), the National Natural Science Foundation of China (nos. 31190060, 81221003, 30730037, 30870834, 91232000), the Program for Changjiang Scholars and Innovative Research Teams in University (PCSIRT) and the Fundamental Research Funds for the Central Universities.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Diffusion profile of Sulforhodamine 101 (SR101) when ejected from the tip of a micropipette.
(A) Schematic of fluorescence detection position. Circles in different colors denote different distances from the tip of the micropipette. Scale bar, 50 μm. (B) Quantitative analysis of SR 101 fluorescence intensities. Fluorescence intensities at three different points on each circle were collected and used for data analysis. Data were normalized to the average fluorescence intensity measured at the position 15 μm from the source after SR101 ejection for 10 min. The arrow marks the time point when the pulsatile application of SR101 was started. Error bars indicate s.e.m. (C) The diffusion profile of SR101 around ∼300 μm from the micropipette tip at different time points is illustrated in a three dimensional surface plot, which was generated using ImageJ with the Interactive 3D Surface Plot plugin. X axis and Y axis indicate distance from the tip of micropipette. Note that the diffusion profile of SR 101 is relatively stable 5 min after its application up to more than 30 min.
Supplementary Figure 2 Double immunofluorescence staining for CD11b (microglial marker) and GFAP (astrocytic marker) in purified primary microglia obtained in Steps 11–13.
Note that most of the cells were CD11b positive and GFAP negative. Scale bar, 20 μm.
Supplementary Figure 1
Diffusion profile of Sulforhodamine 101 (SR101) when ejected from the tip of a micropipette. (PDF 411 kb)
Supplementary Figure 2
Double immunofluorescence staining for CD11b (microglial marker) and GFAP (astrocytic marker) in purified primary microglia obtained in Steps 11–13. (PDF 158 kb)
Microglial migration induced by application of 1 mM ATPγS.
Time-lapse images were acquired by a confocal microscope (Olympus FV1000, Japan) with a 20x air objective (UPLSAPO 20×, 0.75 NA). 1 mM ATPγS was ejected from the micropipette after ten frames (300 s) and frames were acquired every 30 seconds. Scale bar, 50 μm. The video speed is six frames per second. (MOV 1537 kb)
Microglial migration induced by application of 10 mM ATPγS.
Time-lapse images were acquired by a confocal microscope (Olympus FV1000, Japan) with a 20x air objective (UPLSAPO 20×, 0.75 NA). Image frames were acquired every 30 seconds and 10 mM ATPγS was ejected from the micropipette after ten frames (300 s). Note that cells in juxtaposition to the tip do not migrate well. Arrows indicate cells with no obvious directed migration (indicated by the arrowheads) or migrate towards the opposite direction (indicated by the arrow). Scale bars, 50 μm. The video speed is six frames per second. (MOV 1410 kb)
Lysosomal trafficking during microglial migration induced by 1 mM ATPγS.
Microglia lysosomes were labeled with 500 nM LysoTracker Red for 30 min before imaging. The LysoTracker Red is presented as red color overlaid on the bright field images. Time-lapse images were acquired by a confocal microscope (Olympus FV1000, Japan) with a 60×water immersion objective (UPLSAPO 60×W, 1.2 NA). 1 mM ATPγS was ejected from the micropipette after the first frame (20 s) and frames were acquired every 20 seconds. The micropipette is positioned at the lower left corner as indicated by the orange arrow. Scale bar, 10 μm. The video speed is two frames per second. (AVI 2171 kb)
Lysosomal tracks of individual microglia during migration induced by 1 mM ATPγS.
An individual microglia with a magnified field from Supplementary Video 3 (as indicated in Fig. 5A). The trajectories of lysosome were reconstructed using Imaris software (Bitplane). The time course of trajectories are presented as pseudocolor indicated at the lower right corner. Scale bar, 5 μm. The video speed is two frames per second. (AVI 2369 kb)
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Wu, Hj., Liu, Yj., Li, Hq. et al. Analysis of microglial migration by a micropipette assay. Nat Protoc 9, 491–500 (2014). https://doi.org/10.1038/nprot.2014.015
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