实验概要
The ability to culture primary neurons under serum-free conditions facilitates tighter control of neuronal studies. Some serum-free media and supplements allow for the low-density neuronal cultures, which in turn enables the study of individual neurons and synapses. This has not been possible using serum–supplemented media without a feeder layer of glial cells. In serum-supplemented media, glial cells continue to multiply, necessitating the use of cytotoxic mitotic inhibitors. Serum also contains unknown and variable levels of growth factors, hormones, vitamins, and proteins. This chapter details the isolation and culture of neural cells in serum-free media and supplements.
主要试剂
1. Isolating Rat Brain Cells
Hibernate®-E Medium
B-27® Serum-Free Supplement
GlutaMAX™-I
Hibernate®-E Medium, without Ca2
Papain
Neurobasal® Medium
Trypan Blue Stain
Pasteur pipettes
Hemacytometer, cell counter and trypan blue, or the Countess® Automated Cell
Counter
Conical tubes (15-mL, 50-mL)
2. Culturing Embryonic Neurons
Poly-D-lysine hydrobromide
48-well plate or 8-chambered slides
Distilled water
Dulbecco’s Phosphate-Buffered Saline (D-PBS)
Neurobasal® Medium
B-27® Serum-Free Supplement
GlutaMAX™-I
3. Immunocytochemistry
Goat serum
Mouse anti-MAP2 antibody
Rabbit anti-GFAP antibody
Alexa Fluor® 488 goat anti-mouse IgG (H L)
Alexa Fluor® 594 goat anti-rabbit IgG (H L)
4’, 6-diamidino-2-phenylindole, dihydrochloride (DAPI)
ProLong® Gold antifade reagent
EM grade paraformaldehyde
实验步骤
1. Coating Culture Vessels with Poly-D-Lysine
1) Prepare a 2-mg/mL poly-D-lysine stock solution in distilled water.
2) Dilute the poly-D-lysine stock solution 1:40 in D-PBS to prepare a 50 μg/mL working solution (i.e., 125 μL of poly-D-lysine stock solution into 5 mL of D-PBS).
3) Coat the surface of the culture vessel with the working solution of poly-D-lysine (150 μL/cm2, i.e., 100 μL per well for a 48-well plate).
4) Incubate the culture vessel at room temperature for 1 hour.
5) Remove the poly-D-lysine solution and rinse 3 times with distilled water. Make sure to rinse the culture vessel thoroughly, because excess poly-D-lysine can be toxic to the cells.
6) Leave the coated vessels uncovered in the laminar hood until the wells have completely dried. You may use the dry plates immediately or store them at 4°C, wrapped tightly with Parafilm®, for up to one week.
2. Isolating Neurons
1) Dissect cortex or hippocampus pairs from ten E-18 rat embryo brains. Remove all the meninges thoroughly.
2) Collect all the tissue in a conical tube containing Hibernate®-E supplemented with 2% B-27® Serum-Free Supplement and 0.5 mM GlutaMAX™-I at 4°C.
3) Allow the tissue to settle to the bottom of the tube and then carefully remove the supernatant leaving only the tissue covered by the medium.
4) Enzymatically digest the tissue in 4 mL of Hibernate®-E medium without Ca2 containing 2 mg/mL of filter-sterilized papain for 30 minutes at 30°C. Gently shake the tube every 5 minutes.
5) Add 6 mL of complete Hibernate®-E medium to the tube and centrifuge for 5 minutes at 150 × g.
6) Remove the supernatant and resuspend the tissue in 5 mL of complete Hibernate®-E medium by pipetting up and down with a fire-polished glass Pasteur pipette.
7) Let the tube stand undisturbed for 2 minutes to allow for the cell debris (if any) to settle down.
8) Transfer the cells to a new tube leaving behind all the debris.
9) Count the cells using a hemacytometer, cell counter and trypan blue, or the Countess® Automated Cell Counter.
10) Centrifuge the tube for 4 minutes at 200 × g.
11) Remove the supernatant and resuspend the cell pellet in Neurobasal® medium with 2% B-27® Serum-Free Supplement and 0.5 mM GlutaMAX™-I for culturing.
3. Culturing Neurons
1) Plate ~1 × 105 cells per well in poly-D-lysine coated 48-well plate or an 8-chambered slide. Bring the cell suspension volume to 500 μL per well by adding complete Neurobasal® medium.
2) Incubate the cells at 37°C in a humidified atmosphere of 5 % CO2 in air.
3) Feed the cells every third day by aspirating half of the medium from each well and replacing it with fresh medium.
4. Characterizing Neural Cells
1) Preparing Paraformaldehyde Fixing Solution
a. Add PBS to 20 g of EM grade paraformaldehyde, and bring the volume up to 100 mL.
b. Add 0.25 mL of 10 N NaOH and heat the solution at 60°C using a magnetic stirrer until the solution is completely dissolved.
c. Filter the solution through a 0.22-μm filter, and cool on ice. Make sure the pH is 7.5–8.0.
d. Aliquot 2 mL in 15-mL tubes, freeze the tubes on dry ice, and store them at –20°C.
2) Fixing Cells
a. Remove the culture medium and gently rinse the cells without dislodging them twice with D-PBS containing Ca2 and Mg2 .
b. Fix the cells with 4% fresh Paraformaldehyde Fixing Solution (PFA) at room temperature for 15 minutes.
c. Rinse the cells three times with D-PBS containing Ca2 and Mg2 .
d. Permeabilize the cells with 0.3% Triton®-X (diluted in D-PBS with Ca2 and Mg2 ) for 5 minutes at room temperature.
e. Rinse the cells three times with D-PBS containing Ca2 and Mg2 .
3) Staining Cells
a. Incubate cells in 5% goat serum diluted in D-PBS with Ca2 and Mg2 for 60 minutes at room temperature.
b. Remove the 5% goat serum solution and incubate the cells overnight with the primary antibody (Mouse anti-MAP2 at 10 μg/mL and/or Rabbit anti-GFAP at 4 μg/mL) diluted in 5% goat serum at 4°C. Ensure that the cell surfaces are covered uniformly with the antibody solution.
c. Wash the cells three times for 5 minutes with D-PBS containing Ca2 and Mg2 (if using a slide, use a staining dish with a magnetic stirrer).
d. Incubate the cells with fluorescence-labeled secondary antibody (Alexa Fluor® 488 goat‑anti mouse (H L) at 10 μg/mL and/or Alexa Fluor® 594 goat-anti rabbit (H L) at 10 μg/mL) diluted in 5% goat serum solution for 60 minutes at room temperature.
e. Wash the cells three times with D-PBS containing Ca2 and Mg2 . In the last wash, counter-stain the cells with DAPI solution (3 ng/mL) for 10 minutes.
f. Rinse the cells with D-PBS, and if desired, mount using 3 drops of ProLong® Gold antifade reagent per slide and seal it with the cover slip. You may store the slides in the dark at 4°C.
g. Observe the cells under the microscope using filters for FITC, Cy5, and DAPI.
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