1. Cells are in suspension -- they are not dead! To make conditioned media: spin cells to bottom of conical tube (be gentle!!). Save the supernatant in a labeled tube that has the date and is labeled “conditioned medium”. Store in fridge if there is extra.
2. Grow cells at room temperature, no CO2. Passage every 3-4 days. Dilute cells 1:5 each split and use 20% conditioned media. Method:
3. to induce gene expression (i.e. tubulin) add copper sulfate to the media. Cells are Neo resistant. Induce day prior to imaging; to image put on previously prepared ConA coated coverslips. Image 30 min after plating on ConA.
a. Put cells from flask in tube; spin; take off supernatant and save
(this is conditioned medium).
b. Resuspend pellet (gently) in 5 mls media. Take clean flask.
c. Add to clean flask:
1 ml conditioned medium (supernatant)
1 ml of cell suspension
3 mls of fresh media
To make ConA coverslips: Product is C-2631 from sigma
Made it like this: 100mgs, want 0.5 mg/ml. Add 200 mls water, syringe filter. Store at -20 in aliquots.
See my lab book Jan 2006, page 50.
To make medium for growth of drosphila cells: