Etchevers:Notebook/Genomics of hNCC/2009/02/03

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Comparison of N2 to B27 to another supplement for NSC cultures. Bacteria.

When telling Julie Mollet that the hNCC grew pretty well in the Sigma Stemline Neural medium without serum, I noticed that I had not added glutamine (!) or antibiotics, but have been using it pure. And the hNCC look pretty good, even dividing, and are quite confluent in the top seeded two wells in the 24 well plate.

Perhaps re-make some more Rich medium, seed them in (top two wells), and then subject them again to the Neural medium but with the proper supplements. Sigma in the datasheet writes of N-2 supplementation (only seemingly available through Gibco; Sigma sells a "N-1" formulation). Invitrogen makes it hard to find the composition, but here is a list from this publication:

Table 2. Composition of B27, N2, and BIT9500 supplements (and I'm adding in Sigma's "N-1" supplement):

BSA + + + -

Transferrin + + + N-2 has 1 mM, N-1 0.5 mg/ml

Insulin + + + N-2 has 8.61 μM human recombinant, N-1 has 0.5 mg/ml bovine pancreas

Progesterone + + - N-2 has 1 mM, N-1 0.73 μg/ml

Putrescine + + - N-2 has 1 mM, N-1 1.6 mg/ml

Sodium selenite + + - N-2 has 1 mM, N-1 0.5 μg/ml

Biotin + - - (and so on thereafter, all the others only in B27 supplement)

L-carnitine

Corticosterone

Ethanolamine

D()-galactose

Glutathione (reduced)

Linolenic acid

Linoleic acid

Retinyl acetate

Selenium

T3 (triodo-1-thyronine)

DL--tocopherol (vitamine E)

DL--tocopherol acetate

Catalase

Superoxide dismutase


I wonder if since I already add in T3 that perhaps I couldn't just purchase the Retinyl Acetate from Sigma as well as the N2 supplement and be done with it, adding in the vitamin A acetate if I want neural differentation? However, many recent publications use B27 without the vitamin A. Maybe just stick with N-2 and perhaps add in T-3 (with FGF-2/EGF) since that is in common with the Rich culture. But if use retinyl acetate, 0.1 mg/L is a used concentration. "RA was used by every research group with a concentration between 1 and 10 μM in the medium."

Invitrogen writes this on its B27 package insert:

B-27 is an optimized serum substitute developed for low density plating and long-term viability and growth of hippocampal and other CNS neurons. By supplementing NEUROBASAL with B-27 and 0.5 mM L-glutamine Cat. No. 25030); excellent long-term viability of rat embryonic hippocampal neurons has been achieved even after four weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2

This publication gives a 1:1 mix of N2:B27 as -

N2B27 medium (1:1 mix of DMEM/F12 supplemented with 25 μg/ml insulin, 100 μg/ml apo-transferrin, 6 ng/ml progesterone, 16 μg/ml putrescine, 30 nM sodium selenite, 50 μg/ml bovine serum albumin fraction V, and neurobasal medium supplemented with B27 without Vitamin A). All chemicals were from Sigma (St. Louis, MO), except the BSA and B27 media, which were from Gibco.
  • Heather 07:39, 3 February 2009 (EST):

There is sterile 80% glycerol on hand. How much to add to 1.2 mL CaCl2-competent bacteria in order to get a final concentration of 15%? (Aliquot then by 200 μL, as used for today's transformation where I added in 200 ng of diluted plasmids pcDNA3, pGL3 and MmPax6 in pBluescript. These are currently recovering from heat shock.)

Answer: the glycerol is 5.333x too concentrated. Need to dilute with the 1.2 mL. So the % volume of glycerol goes up and the relative % of bacteria goes down in the final dilution.

1.2 mL + 0.21 mL / 1 = 1.41 mL final at 15% glycerol = 1.2/0.85

0.21 mL = 0.262 mL / 0.8 so 1.2 mL bacteria + 0.26 mL 80% glycerol = 1.46 mL final at 15% glycerol. Aliquot by 200 μL.

  • Heather 09:19, 3 February 2009 (EST):
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