Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

All experiments involving live animals described in this protocol are conducted according to the National and International laws and were approved by the local University Animal Care and Use Committee (CEUA/UFRN), under the license 009/2014. The following protocol is performed in a sterile environment. Familiarity with basic cell culture is expected.

1. Dorsolateral Telencephalon Microdissection

1. Prepare the dissection medium (100 mL): 98.5 mL Hank's Balanced Salt Solution (HBSS), 0.5 mL 5 M HEPES pH 7.4, 1 mL penicillin/streptomycin (10,000 units/mL and 10,000 µg/mL).
2. Filter-sterilize the dissection medium.
3. Remove embryonic day 14 (E14) embryos from a pregnant mouse C57/Bl6 (Mus musculus) under anesthesia with isoflurane.
   NOTE: Consider E0 as the day of vaginal plug detection. Dissection should be completed no later than 1 h after removal from the pregnant mouse. This protocol also applies for embryos E11-E17.
4. Remove the embryos by hysterectomy under sterile conditions.
5. Transfer the embryos to a Petri dish with cold dissection medium (4 °C).
6. Remove the brains from the skull by cutting along the longitudinal fissure. The number of embryos usually varies from five to ten. Each E14 brain yields approximately 2 x 10⁶ cells, including progenitors and postmitotic neurons (1:1 ratio)
   NOTE: Use the stereomicroscope for the following steps.
7. Remove the meninges using dissection forceps. Split the telencephalon in the middle to separate the hemispheres. To isolate the dorsolateral telencephalon, cut along the dorsomedial curve and pallial-subpallial boundary using dissection forceps. Transfer the dorsolateral telencephalon to a 2 mL tube with cold dissection medium (4 °C) until all brains are dissected.

2. Cell Dissociation and Plating

1. Prepare the proliferation medium (50 mL of DMEM+10% FCS): 44 mL Dulbecco Modified Eagle's Medium (DMEM), 5mL Fetal Calf Serum (FCS), 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 µg/mL). Filter-sterilize the proliferation medium.
2. Prepare differentiation medium (50 mL of DMEM+2% B27): 48 mL DMEM, 1 mL B27, 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 µg/mL). Filter-sterilize the differentiation medium.
3. After micro-dissection of the dorsolateral telencephalon from E14 mice, centrifuge the tube with the collected tissue and cold dissection medium (for 5 min at 4 °C, 340 x g) to precipitate the tissue.
4. Remove the supernatant with a pipette and add 1 mL of pre-warmed (37 °C) Trypsin-EDTA (0.05%) for chemical digestion. Incubate for 15 min at 37 °C. Add 2 mL of proliferation medium to stop trypsin activity.
5. Polish the tip of a glass Pasteur pipette over a gas burner or Bunsen burner for few seconds to slightly narrow the aperture. Wash the pipette by aspirating FCS to coat the tip. Avoiding bubbles, dissociate mechanically the cells with a fire-polished and FCS-coated Pasteur pipette.
6. Centrifuge the cells (for 5 min at 4 °C, 340 x g). Remove supernatant with a pipette. Add 1 mL of proliferation medium and resuspend the cells using a pipette. Repeat this step once.
7. Prepare a 1:1 dilution of the cell suspension using a 0.4% Trypan Blue solution. Non-viable cells will be blue. Count the number of viable cells (unstained) using a Neubauer chamber.
8. Dilute the cells in proliferation medium to get 10⁶ cells/mL. Add 500 µL of the cell suspension to each well of a 24 well tissue culture plate (approximately 2.5 × 10⁵ cells/cm²). Incubate cells at 37 °C and 5% CO₂.
   NOTE: Do not use glass coverslips, as they can move during the experiment and change the field of observation. Alternatively, use glass bottom tissue culture plates.
   NOTE: If you are not using cell-culture treated multidished, it is important to pre-treat your plates with Poly-D-Lysin (50 µg/ml) 2 h at 37 °C. PDL-treated plates can be stored at 4 °C after washing with distilled water.

3. Retroviral-mediated Transfection

NOTE: This is a simple method to transfect only progenitor cells. However, other viral vectors or chemical/electrical transfection can be used to insert genes of interest in cultured cells.

1. To manipulate gene expression and label cells with fluorescent reporter proteins, add retroviruses carrying plasmids with genes of interest 2 h after cell plating.
   NOTE: Retroviral vectors only integrate into the genome of dividing cells. Therefore, it is important to infect cells when proliferation is high.
   NOTE: We used a plasmid containing the Beta-actin/CMV fusion sequence (CAG) and an internal ribosomal entry site (IRES) followed by the coding sequence for the green fluorescent protein (GFP), designated pCAG-IRES-GFP⁴⁴. Genes of interest could be cloned between the CAG promoter and IRES sequence³⁷.
2. Dilute the retrovirus in differentiation medium to obtain the desired number of particles per mL. Add 500 µL of differentiation medium with retrovirus to each well.
   NOTE: The amount of retroviruses used is dependent on its titer and should be calculated to obtain 50 to 100 infected cells per well. This titer allows the identification of isolated GFP-expressing clones, whose relationship can be further confirmed by tracking phase contrast images (see section 6). The titration of retrovirus should be performed using the same cell population⁴⁵.

4. Time-lapse Video-microscopy

NOTE: This step requires an inverted fluorescence microscope with incubation chamber (see Table of Materials).

1. After the retrovirus infection, place the tissue culture plate into an incubator connected to temperature and CO₂ controllers.
   NOTE: Preheating the chamber for 3 h prior to starting imaging could help minimize drift and defocus during the acquisition.
   NOTE: The chamber should maintain the cells at constant conditions of 37 °C and 5% CO₂.
2. Select a position in the XY axis to serve as a zero-point (XY = 0). This will allow a reference to find the positions again in the future, for instance after post-imaging immunocytochemistry (see section 5).
   NOTE: It is advisable to draw a sign in the plate that can be used as a reference to determine the zero-point.
3. Select the positions to be imaged at 10X magnification.
   NOTE: Select 10-15 positions per well to increase the likelihood to observe retrovirally-transfected cells.
   NOTE: Long-distance high magnification objectives (20X or 40X) can be used to gain higher resolution images. Depending on the working distance of objectives, glass-bottom plates may be required.
4. Acquire phase contrast images every 5 min and fluorescence images every 3 h to decrease phototoxicity. Check and adjust the focus in the first 3 h of experiment, as it may change while the temperature equilibrates. Check and adjust the focus daily.
   NOTE: We observe that the focus becomes stable after the first 24 h. However, it is advisable to monitor the focus. Alternatively, use a software-based autofocus system.
5. After 7 days, stop acquisition and proceed to post-imaging immunocytochemistry (see section 5).
6. After immunocytochemistry, replace the plate in the microscope incubator, find the zero-point, reset XYZ = 0 and reload the experiment with saved positions. Acquire the fluorescence images of each position using appropriate fluorescence filters.

5. Post-imaging Immunocytochemistry

1. After 7 days in culture, fix the cell culture with 4% Paraformaldehyde (PFA) fixative for 15 min at room temperature.
   CAUTION: PFA is toxic and should be handle in a chemical fume hood.
2. Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.
3. Incubate in primary antibodies overnight at 4 °C in 0.5% triton and 10% of normal goat serum in PBS 0.1 M.
   NOTE: Use anti-Microtubule Associated Protein 2 (MAP2) antibody to confirm the neuronal phenotype (mouse IgG1, 1:1,000) and anti-GFP to label the transduced cells (chicken antibody, 1:500). Other antibodies can be used to detect progenitor or glial cells.
4. Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.
5. Dilute secondary antibodies in 0.5% triton and 10% of normal goat serum in PBS 0.1 M and incubate for 2 h at room temperature. Recommended dilution: 1:1,000.
6. Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.
7. For nuclei staining, incubate the cells for 5 min with 0.1 µg/mL 4′6′-diamino-2-phenylindone (DAPI) in PBS 0.1 M.
8. Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.
9. Keep the cells in PBS 0.1 M and protect from light (e.g., wrap the plate with foil).

6. Cell Tracking

NOTE: Here, we briefly describe the main steps to analyze time-lapse video-microscopy data using the software The Tracking Tool (tTt), which is available at the link provided in Table of Materials.

NOTE: If the image acquisition is performed with other software, import the image data with tTt Converter available in the tTt installer (link provided in Table of Materials). Only png, tif or jpg image formats are accepted. Follow the steps shown at Figure 1 A – B after starting the tTt Converter.

1. Start the software tTt and choose a user name. Click on "add user" and "continue" button (Figure 2A). Browse and select the "TTTWorkFolder" (Figure 2B). Lineages trees, statistics and exported images will be saved in this folder. Next, select the experiment to be analyzed and load it (Figure 2B).
2. If the images were converted previously by tTt Converter, click on "LogFileConverter" (Figure 3A) and specify the number of seconds between two consecutive time points in the LogFileConverter window. Click on "Convert log files for whole experiment" (Figure 1C).
3. In a list of images, which appear in the tTt window, select the desired images that by loading manually or using the option displayed in the program. Load images (Figure 3).
4. Select File > Open > New colony. Click on "Tracking", followed by "Start Tracking" to start the tracking in a chosen position.
5. A movie window will appear (Figure 4). Select the cell using the tracking circle and press the key 0. The software will go to the next frame. Keep on positioning the tracking circle around the tracked cell using the mouse and click 0 in each frame to add a track mark on the cell.
6. Use the keys 1 and 3 to move to the previous or next frame, respectively. To delete a track mark, just click on the correct position to mark and press 0.
7. If the cell divided, select the button "division" (red rectangle at Figure 4). Continue tracking one daughter cell by pressing Shift+D. To track the second daughter cell, select it in the cell editor and press F2.
8. If the cell died during the video microscopy, select "apoptosis" (blue rectangle at Figure 4).
   NOTE: Although the software labels the event as "apoptosis", other mechanisms may be responsible for the cell death.
9. If the cell leaves the field of observation or intermingles with neighboring cells precluding tracking, select "lost" (green rectangle at Figure 4).
10. To stop tracking and continue later click on "interrupt" (purple rectangle at Figure 4).
    NOTE: In the Cell editor window a tree will be generated during the tracking. To save the lineage tree, select File > Save > Current tree (in the Cell editor window).
11. To start tracking a different cell, click on the cell using the right mouse button or select the cell using the left mouse button and click on Tracking > Start Tracking.
12. To export the movie with the tracking data, the software should not be in "tracking mode". To turn off the tracking mode, execute step 6.10. In the Movie window select Export > Movie. Set up the format, range of frames, number of frames per second and bitrate. Click on "Start export".

7. Quantifications

NOTE: Several measurements can be performed using video microscopy data⁴⁶. Here, we describe three possibilities that are exemplified later in the "representative results" section.

1. Quantify the cell survival for each cell lineage by dividing the number of cells alive at different time points by the total number of cells generated before these time points were reached within individual clones³⁷.
2. Quantify the proportion of symmetric progenitor (SP, both daughter cells continue to proliferate), asymmetric (A, one daughter cell continues to proliferate and the other becomes postmitotic), or symmetric terminal (ST, both daughter cells become postmitotic)^15,38.
3. Measure the cell cycle length by calculating the time span of proliferating cells between their generation and division¹⁵.