|
This is a cached page for the URL (http://www.stanford.edu/~rnusse/ownpage/protdisccells.html).
To see the most recent version of this page, please click here.
| | Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache | | |
protocols disc cells GENERAL MAINTENANCE INSTRUCTIONS FOR IMAGINAL DISC CELL LINES
This is our lab protocol for growing imaginal disc cells (clone-8)
The Milner Lab website has additional protocols for clone-8 cells.
Clone 8 cells are passaged every 3-4 days from confluent T25 flasks (about 5 x 107 cells): split roughly 1/5 to 1/10. Never allow cultures to become too dense for they will die rapidly! If 95% are dead the day after you split them you have probably let them grow too dense. For the same reason, always use subconfluent cells for biochemistry or RNA isolations. These cells are tightly adherent, especially when they have been passaged recently. Wash the cells with 3ml of trypsin, prior to trypsinization. Trypsinize for about 1 min at room temperature. Tap the flasks real hard to detach cells, immediately add 5 ml of medium to inactivate the trypsin. Spin down cells at 1200rpm in a table top centrifuge and resuspend to required density. Always use a fresh flask when transferring cells.
Cells can be frozen in complete M3 medium + 10% FCS and 10% DMSO; again use subconfluent cultures.
MAKING FLY EXTRACT FOR WING DISC CELL MAINTENANCE
(Cullen and Milner, TISSUE AND CELL, 1991 23(1):29-39.)
Start with a collection of about 30g of healthy flies.
200 flies weigh 0.22g. Use 1.5ml M3 medium per 0.22g; to make 200ml homogenate, use 30g of flies. Place flies in freezer for 20 min.
Place 30g of frozen flies and 200ml of medium into a blender.
"Puree" in blender approximately 2-3 minutes.
Spin mush at 3000 rpm in a table top centrifuge for 20 min.
Remove supernatant (leaving exoskeletons and eye pigment) and transfer to a new tube.
Remove oily layer on top.
Heat inactivate in 60 C waterbath for 30 min. You will see a precipitate form.
Centrifuge at 3000 rpm for 30 min.
Transfer supernatant to new tube and spin again for 15 min.
Remove supernatant and sterilize through a 0.22 um filter.
Store in 12.5 ml aliquots (2.5% final in 500ml) and keep at -20 C.
REAGENTS
The complete M3 medium has the following additives.
2% FBS 2.5% fly extract
5 ug/ml insulin
1/2 x penstrep (100 x bottle = 5000 ug/ml)
Adding insulin.
Insulin (Sigma, I-6634)
Dissolve 5mg insulin in 0.3ml of 0.01N HCl each time medium is made.
Add M3 medium to 5ml.
Add 2.5ml insulin solution to 500ml M3 medium.
Sterilize complete medium through a 0.22um filter. Trypsin
Gibco BRL Trypsin - EDTA .05% Catalog #25300-054 PBS
Sigma #D5927 M3 Medium
Shields and Sangs M3 medium, Sigma (#S3652)
FBS (heat inactivated tested for insect culture), Sigma (#F3018) TRANSFECTIONS
Imaginal wing disc cell lines can be transfected using the conventional Ca++ phosphate coprecipitation method.
Cells should be split 1:4 the day prior to the transfection from a subconfluent T75 flask into 10cm dishes.
Cells are left with the DNA coprecipitate overnight.
The next day the precipitates should be washed away with PBS, trypsinsized and transferred to a clean dish. The cells should be allowed to recover one day before drug selection is started. Transients also work well in these cells. For drug selection 250 ug/ml hygromycin seems to give good results although early on during the selection some drug resistant colonies do appear.
Colonies should appear in about 14 days. When the colonies consist of a few hundred cells they can be scraped off the dish with a sterile yellow tip and transferred to a 12 well dish. Usually after another 2 weeks or so these can be transferred to a T25 flask.