Cell cultures and transfection

PRIMARY CULTURE OF EMBRYONIC RAT NEURONS AND TRANSFECTION

Protocol for primary striated culture 

Day 1:

Prepare 5% poly D-lysine solution: Add 5 ml of poly D-lysine (Millipore, A-003-E, 1mg/ml) to 95 ml sterile water.

1. Add poly D-lysine for mouse tissue culture for rat tissue culture to a 24-well plate (0.5 ml/well). The solution contains poly D-lysine at a final concentration of 0.05 mg/ml. Swirl the plate to ensure that the coating mix covers the entire bottom of the plate and incubate the plate in the cell culture incubator for overnight.

2. Prepare neural growth medium NB+++ 

For 50 ml:

48 ml of neural basal medium (NB) 1 ml of B-27 supplement (50X)

0.5 ml of Glutamax (100X)

0.5 ml of Penicillin/Streptomycin (100X)

3. Prepare OPTI-MEM + Glucose solution 

500 ml OPTI-MEM (Invitrogen)

4 ml of 2.5 M glucose

4. Prepare dissociation medium (DM)

Dissolve following ingredients and make up to 1000 ml with sterile water and then filter sterilize and store it at 4°C. It will have pH 7.5.

5. Prepare 10X Kynurenic acid (KY) solution

Dissolve the following ingredients in sterile water and adjust the pH 7.4 with NaOH and add MgCl₂ and then make up to 1000 ml with sterile water. Sterile filter and store it at 4°C.

Day 2:

1. Wash wells twice with sterile water and dry them in the cell culture incubator.
2. Prepare DM/KY (1:9) and keep it on ice while dissecting the tissue.
3. Prepare trypsin inhibitor (Sigma#T9253-5G): Dissolve a pinch of trypsin inhibitor (~15 mg) in DM/KY solution (color may change) and adjust pH 7.5 to 7.6 with 1 M NaOH (25µl) and then make up to 10 ml with DM/KY (color will be retained to red). Sterile filter (0.2µm) it. It can be stored at RT or on ice while dissecting the tissue.
4. Prepare papain with L-Cysteine (Sigma#c-7880): Dissolve a pinch of L-Cysteine (~20 mg) in DM/KY solution (color may change) and adjust pH 7.5 to 7.6 with 1M NaOH (25 µl) and then make up to 10 ml with DM/KY (Red color will be retained). Then add 75 µl of 30 U/mg papain (Worthington, PAP 100 mg) with a cut tip. Sterile filter (0.2µm) it. It can be kept at RT or on ice while dissecting the tissue. Note: Papain can be added at the end of dissecting cortex or hippocampus.

5. Pour ice-cold DM/KY solution into several culture dishes: 1 large dish for the pups and 10 cm dishes for the pup heads, for the intact brains and for the dissected striatum. Place dishes on ice and pour the entire solution under the hood to keep material sterile.

6. Use autoclaved dissection tools, including several pairs of forceps, chemical spatula and a pair of small and large scissors.
7. Dissection of cortex:
   1. Sacrifice the pregnant rat with CO2/2pts/5-10min. After the rat fails to move spontaneously or in response to pain, puncture each lung with a syringe. Clean the belly with alcohol and then incise along the abdomen and remove the uterus. Place the pups into the large culture dish on ice. Try to avoid the pups contact with mother rat surface. Remove the head from the pups and place heads on a 10 cm culture dish with DM/KY solution on ice. Hereafter, everything will be done on ice under a dissection microscope.
   2. Isolate cortex from the brain and keep it in a 15 ml sterile tube containing DM/KY solution on ice. Note: Keep the papain and trypsin inhibitor at 37°C water bath for 20 min before completing the isolation of tissues.
8. Remove DM/KY and add 10 ml of papain solution to the tissue and then incubate it at 37°C water bath for 15 min. Mix it every 5 min by swirling the tube.
9. Remove papain solution after 20 min incubation and add 10 ml of trypsin inhibitor to the tissue and then continue incubation at 37°C water bath for 15 min. Mix it for every 5min by swirling the tube.
10. Remove trypsin inhibitor and wash the tissue once with 10 ml of OPTI-MEM + Glucose medium in the cell culture hood by allowing the tissue to settle down. Supernatant will be discarded at this step.
11. Trituration: Add 5 ml of OPTI-MEM + Glucose to the tissue and triturate gently with a 5 ml pipette until the solution turns cloudy. After a few times of trituration, collect the supernatant in a 50 ml sterile tube, which can be kept at RT. Then add 5 ml of OPTI-MEM + Glucose to the remaining tissue and triturate a few times and collect the supernatant. If the supernatant is not cloudy, vigorously triturate the tissue by crushing the tissue on wall of the tube and collect the supernatant. It can be done 10 times for 10-15 brains. Tissue debris and DNA will settle down to the bottom of the tube and collect the supernatant, which contains cells.
12. Count number of cells with trypan blue and dilute appropriate cells with OPTI-MEM + Glucose and then plate neurons. Note: ~650,000 to 750,000 cells per well in a 24-well culture plate or 100,000 cells per well in a 96-well plate for survival analysis. 2-4x106 cells in 6-well plates for western blot.
13. Plate the desired number of cells per well and incubate at 37°C for 1 h.
14. Neurons should be attached at the bottom of the well. Carefully aspirate OPTI-MEM + Glucose medium and add (1 ml/well in a 24-well plate) pre-warmed neural growth medium NB+++.
15. Each 3 days, replace 50% medium with fresh neural growth medium NB+++.

Transfection with Lipofectamine 2000

1. Use neurons 4 days after in vitro. This protocol is optimized for cultured neurons in 24-well plates.
2. Prepare OptiMem at RT, and NB/KY solution (9:1), DNA plasmid and Lipofectamine 2000 reagent on ice.
3. Prepare tube A (with DNA) and tube B (with Lipofectamine):
   1. Tube A: 50 ul OptiMem + 1 ug DNA/per well
   2. Tube B: 50 ul OptiMem + 2 ul Lipofectamine/per well
4. Mix gently and incubate Tubes A and B at RT for 5 min.
5. Mix the content of Tube A in to the Tube B.
6. Vortex and incubate at RT for 20 min.
7. Meanwhile, remove media from your cells, wash with 0.5 ml NB/KY solution/well, and add fresh 0.5 ml NB/KY solution /well. Keep cells in NB/KY solution.
8. Add the content of the mix drop by drop in to the well: 100 ul of the mix /well; final volume 600 ul.
9. Incubate cells at 37°C for 2 h. Wash cells with NB/KY solution and add 1 ml of neural growth medium NB+++.

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