Isolation and Transfection of Mouse Primary Neurons

Materials

  • 2x curved forceps
  • 1x 10 cm dish
  • 1x razor blade
  • 4x 1 mL micropipette tips -- 2 mL serological pipettes (or wide bore pipette tips)

Pictured:

A.  1x No. 10 scalpel
B.  2x Dumont No. 5 forceps
C.  1x 22.5° micro knife
D.  1x small flat spatula
E.  2x sterile syringes
F.  2x 27G ½ inch needles
G.  3x 6 cm dish
H.  7x 15 mL conical tubes
I.    1 x microscissors*

*microscissors are required for isolation of mouse embryonic hippocampus

Media and Solutions

-- 10X TrypLE™ Select: (A12177)
-- UltraPure™ DNase/
    RNase Free Distilled
    Water: (10977015)
-- HEPES, 1M Buffer Solution: (15630106)
-- B-27® Serum-Free Supplement (50X): (17504044)
-- GlutaMAX™: (35050061)
-- Neurobasal® Media: (21103-049)

 

 Mouse Primary Neurons-  Materials

 

Isolation Setup

Coating of Tissue Culture Dishes   Coating of Tissue Culture Dishes

Prepare one day prior to isolation under sterile conditions.

 

A.  Start with Poly-D-Lysine (BD cat no. 354210) 20 mg vial; reconstitute with 4 mL UltraPure™ Distilled water to 5mg/ml stock solution. Divide to 1 mL aliquots and store at -20°C.

B.  Dilute stock to working solution of 0.05 mg/ml. Add 1 mL of working solution to 35 mm dishes and incubate for 2 hours.

C.  Wash 3x with UltraPure™ Distilled water. Let air dry for ~4 hours.

D.  Parafilm wrap and store at 4°C for up to 2 weeks.

 

Coating of Tissue Culture Dishes     Preparing Dissection Solution

Adapted from: “Preparation of Dissociated Mouse Cortical Neuron Cultures.” Lutz G. W. Hilgenberg, Martin A. Smith. J Vis Exp. 2007; (10): 562. Published online 2007 December 19, doi: 10.3791/562. PMCID: PMC2557074.

 A. Make Solution A by adding the following materials to 100 mL UltraPure™ Distilled water. Bring final volume to 500 mL. Filter and store at 4°C.

Raw Material Amount Final Concentration
Sodium Chloride (NaCl)80.0 g137 mM
Potassium Chloride (KCl)4.0 g5.4 mM
Sodium Phosphate Dibasic Anhydrous (Na2HPO4)0.24 g0.17 mM
Potassium Phosphate Monobasic Anhydrous (KH2PO4)0.3 g0.22 mM

B. Make solution B by diluting 2.5ml of 1M HEPES Buffer Solution in 247.5ml of UltraPure™ Distilled water to a final working concentration of 9.9mM. Filter and store at 4°C.

C. Complete Dissection Solution (DS) by adding the following materials to 400 mL UltraPure™ Distilled water. Adjust pH to 7.4 with NaOH. Bring final volume to 500 mL. Filter and store at 4°C.

Raw Material Amount Final Concentration
Solution A25 mL--
Solution B4.0 g14 mL--
D-Glucose3.0 g33.3 mM
Sucrose7.5 g43.8 mM

 

Coating of Tissue Culture Dishes  Preparing Complete Neurobasal Media

Add 10 mL of B-27® Serum-Free Supplement (50X) and 1.25 mL of 200 mM GlutaMAX™ to one 500 mL bottle of Neurobasal® Media.

 

Coating of Tissue Culture Dishes  Preparing Trypsin Inhibitor / BSA Stock Solutions

Dissolve 1 g BSA (SIGMA, A-7030) and 1 g Trypsin Inhibitor (Worthington, LS003087) in 20 mL prepared Dissection Solution. Adjust pH to 7.4 with 1 N NaOH. Sterile filter through 0.2 μm syringe filter. Divide into 800 μL aliquots and store at -20°C.

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Isolation

Coating of Tissue Culture Dishes    

Sacrifice an appropriately timed pregnant female (16.5 days) according to your facilities’ IACUC regulations. Open abdominal cavity and remove uterus, containing embryos. Place in 10 cm dish containing cold dissection solution.

 

Coating of Tissue Culture Dishes  

Carefully remove placenta using two small curved forceps with gentle opposite pulling motions. Then remove embryo from remaining sac and place in fresh cold dissection solution. Note: If isolating tissue from multiple embryos, proceed to isolate all required embryos from the uterus and the embryonic sac and keep submerged in cold dissection solution on ice

Coating of Tissue Culture Dishes   Carefully decapitate animal with clean sterile scalpel and place in 6 cm dish containing 4-5 mL dissection solution.

 

Coating of Tissue Culture Dishes   Under dissecting microscope, use two No.5 Dumont forceps and carefully remove thin skin layer by pinching skin at center and peeling away. To cut and remove forming skull pieces, anchor head by piercing orbital cavities with forceps and use a 22.5° angled micro knife to make a very shallow incision from anterior to posterior.

 

isolation of mouse primary neurons
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Coating of Tissue Culture Dishes   Without removing the anchoring forceps, carefully place the second set of forceps at an angle to grab the skull flap without piercing the tissue. Pull gently to remove soft bone fragments. Try to avoid pulling off small pieces with jerky movements.

 

isolation of mouse primary neurons
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Coating of Tissue Culture Dishes   Using small flat spatula, angled beneath the brain, lift carefully, remove brain from skull and place in a clean 6 cm dish with fresh dissection solution.

Coating of Tissue Culture Dishes
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Coating of Tissue Culture Dishes   Separate two hemispheres from the midbrain. Using one sterile syringe with needle attached, anchor tissue in the midbrain and use other needle to gently separate the two hemispheres along the middle of the sagittal plane. Then use the needles to separate the midbrain from each of the two hemispheres.

 

isolation of mouse primary neurons
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Coating of Tissue Culture Dishes   With the outer surface of the hemisphere facing upwards, remove meninges. Use one needle to anchor tissue and forceps to carefully peel away meninges from the outer surface. Without releasing meninges from forceps, turn tissue over, and carefully peel away remaining meninges. If done properly, entire meninges can be removed in one piece.

 

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Coating of Tissue Culture Dishes   Proceed to isolate cortex from remaining inner midbrain region for each hemisphere. Using one needle as an anchor, carefully remove the midbrain from the inner side of the cortex with short cuts using the other needle. If isolating hippocampus,anchor tissue and use microdissection scissors to isolate from cortices.


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Coating of Tissue Culture Dishes   Once desired regions have been isolated, the cortices need to be cut into approximately 5-6 smaller pieces for digestion. To cut cortices, use an x-position with the needles, where one needle serves as an anchor while the other needle is used to cut tissue. The hippocampus can remain whole. Note: If isolating tissue from multiple brains, isolated tissue can be stored in dissection solution on ice for up to 30 minutes.

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Coating of Tissue Culture Dishes   Using syringe with no needle 11 attachment, or wide bore micropipette tip, aspirate tissue pieces and place in clean dish. Whichever method used, take special care as to not disrupt or tear tissue pieces.

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Coating of Tissue Culture Dishes   Add 5 mL sterile 10X TrypLE™ Select and place dish in 37°C incubator for 25-30 minutes.

 

Coating of Tissue Culture Dishes   During incubation time prepare wash solutions and media if have not done so already. Keep wash solutions on ice. Ensure complete media is pre-warmed to 37°C prior to use.

 

Solutions High Wash (Hi) Low Wash (Li)
Trypsin Inhibitor/ BSA Stock 600 μL 160 μL
Dissection Solution 2.4 mL 7.84 mL
Aliquot Volume 1.5 mL 2.6 mL
Aliquot # Per Wash 2 3

 

Coating of Tissue Culture Dishes   Note: All subseqent steps are performed in a laminar flow hood using either a 2ml serological pipette or wide bore pipette tip for aspiration. Remove dish containing tissue pieces from 37°C and aspirate tissue pieces with as little TrypLE™ solution as possible. Place in 15 mL conical tube containing 10 mL cold dissection solution and let tissue settle for 2 minutes.

  

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Coating of Tissue Culture Dishes   Aspirate tissue pieces with as little solution as possible, and transfer to 1st High Trypsin inhibitor wash (Hi), swirl and let tissue settle for 2 minutes. Repeat for 2nd Hi wash.

 
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Coating of Tissue Culture Dishes   Aspirate tissue pieces with as little solution 16 as possible, and transfer to 1st Low Trypsin inhibitor wash (Li), swirl and let tissue settle for 2 minutes. Repeat for 2nd and 3rd Li washes.

 
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Coating of Tissue Culture Dishes   After the last Li wash, aspirate tissue pieces and place in 6 cm dish containing 5 mL prewarmed complete media.

  

Coating of Tissue Culture Dishes   With a clean razor blade cut the tips off of three 1 mL micropipette tips to give 3 different bore sizes of tips for trituration.

 

 

Coating of Tissue Culture Dishes   Starting with the widest bore size tip and subsequently moving to the next smaller size, slowly triturate tissue pieces by pipetting up and down 10-20 times. Switch to uncut tip for the final triuturation. All trituration should take less than 5 minutes.

  

Coating of Tissue Culture Dishes   Transfer media containing cells to clean 15 mL conical. Rinse dish with 5 mL additional complete media and add to conical tube for final volume of 10 mL.

  

Coating of Tissue Culture Dishes   Allow larger tissue pieces to settle for approximately 2 minutes and then transfer 9.5 mL of cell suspension, taking care not to disturb settled tissue at bottom of tube, to a new, clean 15 mL conical.

 

Coating of Tissue Culture Dishes   Perform Trypan Blue exclusion assay to count cells and assess viability. Plate at 200K-300K per plate for best transfection results.

 

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Immunostaining to Characterize Neurons

Reagents Required:

-- 1X D-PBS containing Ca2+ and Mg2+: (14040-141)
-- Goat Serum: (16210-064)
-- Mouse Anti-MAP2 antibody: (13-1500)
-- Rabbit Anti-GFAP antibody
-- Alexa Fluor®488 goat anti-mouse: (A-11029)
-- Alexa Fluor®594 goat anti-rabbit: (A-11037)
-- 4’, 6-diamidino-2-phenylindole, dihydrochloride (DAPI): (D1306)
-- 1% Triton®X-100: (HFH10)
-- ProLong® Gold Antifade Reagent: (P36930)
-- EM Grade Paraformaldehyde (PFA): (Electron Microscopy Services)

Coating of Tissue Culture Dishes    

Preparing 20% PFA Stock Solution

 

 A.  Add 1X PBS to 20g of PFA, and bring 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 0.2 μm filter and cool on ice. Ensure the pH remains around 7.5-8.0.

 D. Aliquot 2 mL into 15 mL tubes, freeze on dry ice, and store at -20°C.

 

Coating of Tissue Culture Dishes      

Preparing 4% PFA For Fixing

Add 8 mL of 1X PBS to one 15 mL tube containing 2 mL of 20% PFA and thaw in 37°C water bath. Cool dissolved solution on ice.

 

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Fixing Cells

Coating of Tissue Culture Dishes   

Remove culture medium and gently rinse cells twice with D-PBS without dislodging them.

 

Coating of Tissue Culture Dishes   

Fix cells with freshly prepared 4% PFA at room temperature for 15 minutes.

                               

Coating of Tissue Culture Dishes   

Rinse cells three times with D-PBS without dislodging them.

 

Coating of Tissue Culture Dishes   

 Permeabilize the cells with 0.3% Triton®X-100 (diluted in D-PBS) for 5 minutes at room temperature.

 

Coating of Tissue Culture Dishes   

Rinse cells three times with D-PBS.

 

Staining & Characterization

Coating of Tissue Culture Dishes    

Incubate cells in 5% goat serum diluted in D-PBS for 60 minutes at room temperature.

 

Coating of Tissue Culture Dishes      

Remove 5% goat serum solution and incubate the cells overnight at 4°C with the primary antibody (Mouse anti-MAP2 at 10 μg/mL and/or Rabbit anti-GFAP) diluted in 5% goat serum.

 

Coating of Tissue Culture Dishes      

Wash cells three times for 5 minutes with D-PBS (if using a slide, use a staining dish with a magnetic stirrer).

 

Coating of Tissue Culture Dishes        

Incubate cells for 60 minutes at room temperature with flourescence-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.

 

Coating of Tissue Culture Dishes     

Wash cells three times with D-PBS. In final wash, counter-stain cells for 10 minutes with DAPI solution (3 ng/mL).

 
Coating of Tissue Culture Dishes     

Rinse cells with D-PBS, and if desired, mount using 3 drops of ProLong® Gold antifade reagent per slide and seal with cover slip. Slides may be stored away from direct light at 4°C.

 

Coating of Tissue Culture Dishes   

Observe cells under microscope using filters for FITC, Cy5, and DAPI.

 

Expected Results

expected results


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Above: Fresh primary Rat Cortical neurons. Immunofluorescence detection of primary neuronal cells stained with mouse anti-MAP2 antibody (green) and presence of astrocytes as detected by rabbit anti-GFAP marker (red). Nuclei are stained with DAPI (blue).

Transfection

Transfect cells according to the following diagram. Volumes are given on a per-plate basis for a 35 mm glass-inserted plate (MatTek, P35G-0-14-C).

transfection
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*mRNA can be generated using Ambion® mMessage mMachine® T7 Ultra Kit (AM1345)

 

Reagents Required    
13778-075Lipofectamine® RNAiMAX Transfection Reagent0.75 ml
11668-027Lipofectamine® 2000 Transfection Reagent0.75 ml

Results

 

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LT176  rev. 06-21-2013