Transfection & Binding Assay Protocol
Plating
(Before starting, you will want to pre-warm your Trypsin/Versene, DMEM,
and DMEM+FCS. Make sure that you have enough of these solutions made and
warmed up before you start.)
1. Take COS-1 flask out of incubator, remove solution by aspiration.
2. Add 10 mL prewarmed (37C) trypsin/versene to flask.
3. Incubate for 10 minutes at 5% CO2).
4. Add 10% fetal calf serum (FCS) to DMEM (25:225 ml) (if necessary).
5. Take the flask from the incubator.
6. Disperse the cells using an electric pipettor and a disposable
10 mL pipette (up and down 10 times).
7. Remove the dispersed cells to a 15 mL red-capped Sarstedt tube.
8. Centrifuge at 3000 rpm for 3 minutes.
9. Remove the supernatant by aspiration (note the pellet volume).
10. Add 12 mL DMEM(+FCS) to the tube with the cell pellet.
11. Disperse cells by pipetting up and down with 10 mL pipette.
12. Label plates.
13. You may use the 12 mL of cell suspension to plate up to 10 plates per
confluent flask. In the case of 10 plates, add 5 mL of cell suspension to
45 mL of DMEM in a 50 mL red-capped Sarstedt tube. Mix this
suspension by inversion. Remove 10 mL to each of 5 labelled plates. Repeat
with another 5 mL of the cell suspension. This will yield 10 plates and 2
mL of suspension. Take the suspension and add it to a flask containing 20
mL of DMEM(+FCS). So from one confluent flask you will get 10 plates and a
flask that is a 6:1 dilution of a confluent flask. If your cell pellet is
smaller, then you may not be able to plate as many plates, or have to
plate them with less cells.
Transfection
1. Add 25 µl of 1µg/µl DNA solution containing the plamid(s) of
interest,
50 µL 2.5 M CaCl2, 425 µl H2O, 500 µl 2X BBS
(pH=6.95) to tubes, let sit at room temp for 30 minutes.
2. Spread drops around on corresponding labeled plates.
3. Place plates in 3% CO2 incubator for 18-24 h.
4. Remove liquid by aspiration.
5. Add 5 ml versene into each plate and let sit 3 minutes.
6. Remove liquid by aspiration.
7. Add 5 ml DMEM (without FCS) to each plate.
8. Remove liquid by aspiration.
9. Add 10 ml DMEM (+10% FCS).
10. Incubate for 48 hours in 5% CO2 incubator.
Preparing for Binding Assay:
~Before removing plates from incubator prepare ligands for binding assay:
~make up 4 L of Tris for harvester, put in freezer
~set out 2 rows of 9 tubes for each receptor
~aliquot 20 µl of .5 nM radioactive (hot) ligand into each tube (need
~to dilute to proper concentration)
~take out stock of 10-3 M cold ligand, adjust volume to 1000µl w/
~Tris, disperse (for peptides add 1/6 volume of proteinase)
~set out 8 other tubes, put 800 µl of Tris in each
~take 200 µl from ligand tube, put in 1st of 8 Tris tubes, disperse
~take 200 µl from this, put in 2nd Tris tube, disperse
~continue until 8th tube has 1000 µl of dispersed Tris + ligand
~aliquot 30 µl of highest concentration cold ligand into the 1st tube
of each row of empty tubes (use continuous Pipetman)
~aliquot 30 µl from 2nd highest concentration ligand tube into 2nd
~tube of each row, continue down the line
~keep on ice until ready to mix w/ homogenate
Harvesting the cells:
Remove plates from incubator.
suck liquid off plates, add 5 ml 50mM Tris to each plate.
scrape cells off bottom of plate w/ rubber policeman.
transfer into tubes, centrifuge.
suck off liquid, add 4 ml Tris, homogenize with VerTishear.
(spin again w/Tris at 12,000 rpm for 20 min if ligand is a peptide).
(suck off solution, add 4 ml Tris, mix w/ continuous pipetman).
aliquot 200 µl of this into each tube of hot and cold ligands, keep
cold.
incubate about 2hr @ room temp.
prep counting vials by adding 10 ml of scintilating sol. to each.
take 2 Tris flasks and tubes out of cold room, pour Tris in Cell Harvester
reservoir, make sure tubes are kept cold.
clean harvester with H2O, flush old Tris from tubes.
wet a filter in Tris, place in harvester.
place rods from harvester into each tube, add Tris, Remove liquid.
add tris again, remove liquid.
when machine is finished (30 sec.) turn off, lift top and take
filter-sheet.
punch a filter into each vials, throw out empty tubes and paper.
wet new filter sheet in H2O before placing in machine.
turn on machine, repeat process.
break-up membranes in counting vials (hit carton hard, repeatedly).
put bottles in counter (put Protocol #9 label on 1st tray).
Press F2 to start counting.