Assessing Interneuron Response to Chemoattractive Cues from Periventricular Endothelial Cells

Abstract

Source: Datta, D., et al. Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons. J. Vis. Exp. (2020).

This video shows a method to test interneuron migration in response to chemoattractive cues from periventricular endothelial cells (PVECs). Interneurons are placed in a central compartment, with PVECs and control cells on either side. Increased migration towards PVECs indicates a positive response to their chemoattractive signals.

Protocol

1. Chemo-attraction Assay

1. Place a three-well culture insert in the center of a poly-L-ornithine/laminin-coated 35 mm dish using sterile tweezers.
2. Turn the dish upside down. Mark the boundary around the middle compartment of the insert using a permanent black marker with an ultra-fine tip.
3. Seed 3 x 10⁴ human GABAergic interneurons in the middle compartment in 70 µL of neuronal medium (Figure 1A).
4. Seed 10⁴ human periventricular endothelial cells in 70 µL of periventricular endothelial cell medium and 10⁴ control endothelial cells in 70 µL of control endothelial cell medium in the two outer compartments, respectively (Figure 1A).
5. Add 1 mL of co-culture medium (50% periventricular maintenance medium without GABA and 50% neuronal medium) along the side of the dish to prevent the coating on the dish from drying.
6. Check under a microscope to verify that cells are not leaking from the insert compartment.
7. Incubate cells for 24 h at 37 °C and 5% CO₂. After 24 h incubation, check under a microscope to verify that cells have attached properly and that there is no leak.
8. After 48 h of seeding, gently remove the insert using a sterile tweezer. Check under the microscope to verify that the cell layer is not disturbed (day 0).
9. Remove the medium and add 1 mL of fresh co-culture medium.
   NOTE: Set aside a required number of dishes for day 0 images.
10. Incubate cells for 36 h at 37 °C and 5% CO₂. After 36 h, aspirate medium, fix cells with 4% PFA for 10 min, and wash 3x with 1x PBS.
11. Stain human GABAergic interneurons with anti-human β-Tubulin or anti-human MAP2 antibody. At the end of the staining procedure, add 1 mL of antifade mounting medium to each dish.

2. Imaging and Data Analysis

1. Place the immuno-stained assay dish under a microscope at 4X magnification.
2. Keep one long edge of the rectangular boundary (made in step 5.10 above) in the field of view. Take images of cells in the cell-free space adjacent to that boundary. Acquire images along the right long edge and the left long edge of the rectangular boundary.
   NOTE: Cells positioned diagonally with respect to the rectangle are not considered due to ambiguity in selecting the short- or long-edge as the starting mark. Also, numbers of cells migrating across the short-edge are often significantly fewer (possibly due to lesser number of starting cells along the short-edge) and are not considered.
3. Open the images in ImageJ. Calculate the distance between each cell and the boundary mark using ImageJ.
4. To assess migration in terms of cell numbers, set a specific distance from the boundary in the acquired image in ImageJ. Count the number of cells that are present within this distance. Calculate average number, standard devia
5. tion, and statistical significance using appropriate software.

Results

Figure 1: Chemo-attraction assay. (A) Schema of the chemo-attraction assay. Using a three-well culture insert, interneurons (IN) were seeded in the middle (green dotted rectangle), while periventricular endothelial cells (PV ECs; orange dotted rec...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Control human endothelial cells	Cellular Dynamics	R1022
Control endothelial Cells Medium Supplement	Cellular Dynamics	M1019
DMEMF/12 medium	Thermofisher Scientific	11320033
E6 medium	Thermofisher Scientific	A1516401
FGF2	Thermofisher Scientific	PHG0261
Fibronectin	Thermofisher Scientific	33016-015
GABA	Sigma-Aldrich	A2129
Hemacytometer	Sigma-Aldrich	Z359629
Human GABAergic neurons	Cellular Dynamics	R1013
Human GABAergic neurons base medium	Cellular Dynamics	M1010
Human GABAergic neuron Neural supplement	Cellular Dynamics	M1032
Laminin	Sigma	L2020
Mounting Medium	Vector laboratories	H-1200
poly-L-ornithin	Sigma	p4957
PBS	Thermofisher Scientific	14190
VEGF-A	Peprotech	100-20
VascuLife VEGF Medium Complete Kit	Lifeline Cell Technologies	LL-0003	Component of control human endothelial cell medium
3-well silicone culture-Insert	ibidi	80369
35 mm dish	Corning	430165
4% PFA solution	Fisher Scientific	AAJ19943K2
Inverted phase contrast microscope	Zeiss	Zeiss Axiovert 40C
Fluorescent microscope	Olympus	FSX-100
Anti-human β-Tubulin antibody	Biolegend	802001
Anti- MAP2 antibody	Neuromics	CH22103