Neuroprogenitor proliferation inNPGrow
Please find enclosed one 2 ml tube containing embryonic day 18 Sprague/Dawley in 2 ml B27/HibernateE. Tissue can be stored at 4-8oC for one week. Also find 12 ml NPGrow (all components are available through Invitrogen Corp.) culture medium.
Needed Supplies:
Ultra-low adhesion 6-well plates (Fisher Scientific)
Trypan Blue (sigma)
Papain (Worthington)[Optional]
Hibernate E (BrainBits, LLC #HE)
Hibernate E-Ca (BrainBits, LLC #HE-Ca) [Optional]
Additional Grwoth Medium: NPGrow
Papain and HE-Ca are optional for maximum yields (step 2B)
Media: NbActiv4-Ra + EGF + FGF(BrainBits, LLC, upon request)
2ml rubber latex bulbs
Isolation, for optional papain method:
1. From the tube with the brain tissue, remove 1 ml medium and save for step 3, being careful not to remove the tissue.
2. Triturate Tissue: Choose Method A) (purely mechanical) or B) (enzymatic)
A) With 1 ml pipettor with sterile blue plastic tip, or silanized 9 inch pasteur pipette with tip barely fire polished (preferable), suck the tissue with medium into the pipette and immediately dispense the contents back into the same container. Take care not to create bubbles. Repeat this trituration step about 5- 10 times or until 90% of tissue is dispersed.
Enzymatic Dissociation:
B) Higher survival can be obtained by incubating the tissue in papain.
i) Transfer brain tissue to 2ml of 2 mg/ml papain (Worthington) in Hibernate E-Calcium without B27, saving original Hibernate E/B27 for step iii.
ii) Incubate with shaking for 10 minutes at 30 C.
iii) Transfer the tissue back to the original HibernateE/B27 with as little Papain as possible.
iv) Triturate as in step 2A.
3. Add back 1 ml medium that you removed in step 1 and mix.
4. Let undispersed pieces settle by gravity for 1 min.
5. Transfer supernatant with cells to a new sterile 15 ml tube.
6. Spin 1100rpm (200 x G), 1 min. Discard supernatant.
7. Flick the tube to disperse the pellet of cells.
8. Resuspend pellet in 4 ml room temperature NPGrow.
9. Aliquot 20 ul and mix with 20 ul 0.4% trypan blue.
10. Count in hemacytometer: calculate cells/ml. Phase bright.
11. Dilute cells with NPGrow(provided) to 8000/ml x ____ml desired (2ml/10cm2 well).
12. Plate 2 mL of cells/10cm2 well, at a concentration of 800 cells/ml (800 cells/cm2 x 10cm2 = 8000 cells/ml x 2ml = 16000 cells/well/2ml).
Incubate 37oC, 5% CO2, 9% oxygen (20% oxygen is O.K.) After 2 days or longer, neuroprogenitors are present. If further culture is desired, change one half of the medium with fresh, warm NPGrow. Change one half every 3 or 4 days. Cells are not attached, so tilt the plate gently and aspirate half media from bottom corner on the top of the media as the cells have settled to the bottom and add fresh media. You may lose cells during media exchange. Otherwise grow out to 5-10 days and observe.