Semisolid Medium For HL 60 cells - (May/04/2009 )

Hello,

i would like to grow HL 60 cells on semisolid medium
i dont know which media i should use..
if anybody does have a protocol ..please let me know

Thank you in advance

Swarna

swarna on May 4 2009, 11:23 AM said:

I assume that you want to grow the cells IN semisolid medium in order to obtain colonies.

A pretty standard method is to:

1. Prepare the normal growth medium (say RPMI 10% FCS) and warm to 37 oC in a waterbath.
2. Prepare 3.6% low melting point agarose (eg Seaplaque from FMC) in water. Autoclave. Before use, melt in boiling water and cool to 37 oC in waterbath.

3. Add 1 ml agarose solution (use a sterile syringe) to 9 ml warm media, mix and return to waterbath.
4. Add cells (depending on cloning efficency say 30 - 100 cells per ml). You will need to find the optimal number that gives well isolated colonies.

5. Plate 1ml cell mix/35mm petri dish.

6. Place dishes at 4 oC for 3 - 5 minutes (on a levels surface) until agarose has set.

7. Incubate 5 - 7 days.

You can used "normal" agarose, but this needs to be maintained at 56 oC and added to the 37 oC medium immediately before plating. You have to work VERY fast. The low melt agarose is easier by far.

Hope this helps.

Hey,

Thank you for ur help
but iam looking for media with methylcellulose..

swarna on May 5 2009, 10:27 AM said:

No problem. But it would be a good idea to state your requirements more carefully.

Using methocel is a pain in the neck. You can buy it as a ready to use liquid but it is very expensive.

A major problem is that it cannot be autoclaved in solution and is difficult to handle. It also has the strange property of dissolving only when cooled. It needs to be prepared very carefully and takes quite an effort:

Methocellulose (x2)

1) Sterilize 500mL of water in a 2L conical flask containing a large magnetic stir bar.

2) Sterilize 24g of 4000 centipoises methylcellulose (#M0512, Sigma) in a 500 ml beaker.

3) Carefully remove the flask containing the water from the autoclave while still hot. Place flask on a heated stir plate inside a laminar flow hood. Bring the water to a boil and slowly (over 15 - 30 minutes) add methylcellulose powder with stirring. Exercise caution as addition of the methylcellulose will cause the boiling water to foam.

4) Allow the methylcellulose solution to cool to approx 37 0C, and add 2X concentrated tissue culture medium. Stir at 4°C overnight.
Aliquot the 2X methylcellulose by pouring approximately 40mL into 50mL polypropylene tubes. Freeze at -20°C.


For use, dilute x2 methylcellulose with FCS/media supplements/x1 basal media. Vortex. Add cells and mix by pumping up-and-down in a syringe. Allow to stand to remove bubbles. Dispense 1ml mix into 35 mm dishes.

Hope this helps, and good luck!

Hey

thank you for your help
It really worked

Thank you Again