Primary cell culture recipes
provided by the great Jung Choi,
and part of science-related notes.
Note: This page describes the procedures used by Jung Choi
on 2/12/04. Most of these have been arrived at after considerable
testing and trial and error. YMMV.
- Glass Coverslips: German 12mm (Electron microscopy sciences, Cat. # 72196-12)
- Wash - use sonicator
- In ethanol (95% +) for 1 hr; rinse with dd-H2O. Repeat.
- In dd-H2O - 30min (3x)
- Separate and dry. (Separating: use tweezers to separate coverslips
in water; take out one by one.) (Drying - a rack of
for coverslips is ideal; if not available, may do
it on wrinkled foil)
- Silicon dish:
- Drill a hole in the bottom (3/8 - 1/2" diameter) of 35mm culture dish.
- Cut a piece of silicon and carefully glue (silicon rubber) onto the bottom of the dish so that the shiny part is shown through the opening. Let it dry overnight
- Wash the silicon surface with soap and water.
- Fill up the dish with ethanol and leave it in for 15min.
- Rinse with dd-H2O. Dry in the hood before applying substrate.
- Enzyme solution
- 50µl of cysteine stock (40mg/ml)
- 100µl EDTA stock (50mM)
- 150µl of 100mM CaCl2 (there is 1M CaCl2 stock too)
- 200 units of Papain
- 10ml HBSS
- 50µl of Dnase stock (0.2mg/ml) - add just before digestion then filter.
- Modified HBSS Soln ("HBSS")
- 0.35g NaHCO3
- 1.3g HEPES
- HBSS (Sigma H2387) in 1L H2O.
- Culture Medium
| ||a. ||Basal Medium Eagle (Invitrogen) || 430ml |
| ||b. ||Glucose ||1.8g |
| ||c. ||Na-Pyruvate (100mM, Invitrogen) || 5ml |
| ||d. ||FBS (Hyclone) || 50ml |
| ||e. ||Mito serum extender (Invitrogen) || 500µl |
| ||f. ||B27 (Invitrogen) || 10ml |
| ||g. ||1 M HEPES || 5ml |
- Substrate (a or b)
- Collagen/PDL mix - for Post-natal island cultures
- 4ml DD-H2O
- 2ml 17mM Acetic Acid
- Rat tail collagen (BD Scientific) - 3.6mg
- 40µl of PDL stock soln (1mg/ml)
- Matrigel (BD biociences, 356234) - micro dots - for embryonic
- Make 1:100 dilution - keep it in ice!!!
- Spray using atomizer
- 24 well plates - use inner 8 wells only and fill up the outer ones with water
- If needed, apply 100µl of collagen/PDL mix onto coverslip or silicon. Incubate during the dissection. For embryonic, use matrigel (see materials section)
- Prepare the enzyme solution.
- Perform dissection (E18 or P1-3); keep tissues in HBSS
- Collect Hippocampus, transfer to 15ml conical tube
- Wash 3x with HBSS. Add Dnase to enzyme solution.
- Filter and add enzyme solution to the tube, then incubate 45 - 50 min.
- Wash 3x with Culture medium
- Add 1-2ml of Conditioned culture medium and mechanically dissociate using 1ml pipette. Be gentle.
- Count the cells. (# x 10K / ml)
- Make appropriate dilutions for desired density with the conditioned culture medium
- Embryonic - 15k/well
- Postnatal neurons on glia - 10k/well
- Postnatal neurons on silicon dish - 50~75k/ml x 2ml
- Glial islands preparation - 5k/well
- Add 500µl to each inner well. Fill up the outer wells with water to minimize evaporation
- Prepare 24µM Ara-C soln (Sigma C6645) in conditioned culture medium. Leave the medium in the incubator for at least half an hour with the cap loose.
- Feeding Schedule:
- Glial islands
- One day after the culture, add 100µl of feeding medium to each well. Monitor the size of the islands throughout the week and add Ara-C as needed. If the neurons remain in the culture after the 2nd week, put the plate in the refrigerator overnight. Islands will be used on 3rd or 4th week.
- Postnatal neurons on glial islands
- Regardless of the growth, add feeding medium the day after. No feeding needed until the recording.
Postnatal Neurons on silicon: after 2nd day of culture, add 400µl of feeding medium. Replace ½ medium with 4µM Ara-C feeding medium weekly.
- Embryonic neurons
- Wait until the neurons and glia cells have grown to form islands (usually 3-4 days, on matrigel is quicker). Then add 100µl. No more feeding needed.
- Alternate method: if the culture has too much debris, prepare 4µM-Ara-C solution in conditioned culture medium and replace the entire (~90%) medium with it. Works just as good.
- Prepare cells at 10k/ml in culture medium and place 20ml in a culture flask (BD353136)
- Replace the medium with 4µM Ara-C containing medium when glias formed smooth islands that cover roughly ½ of area.
- In 2-3 weeks most neurons should die. If not, incubate in the refrigerator overnight.
- When the glias are neuron-free, they are ready to be used to condition the medium
- Simply incubate fresh medium overnight and use it the next day. When not in use, incubate them in 4µM Ara-C culture medium. Glias are good up to 4 months, but are generally replaced every 1-2 months.