Tissue Culture
To thaw frozen cells:
1) take tube of frozen cells from the liquid nitrogen container (waterman lab), place on dry ice
2) bring medium up to 37C in water bath, then thaw the frozen cells in the water bath
3) add 8 mL of medium without/with serum to a 15 mL centrifuge tube
4) add thawed cells to 15 mL centrifuge tube w/ medium
5) rinse the original tube to transfer all cells
6) centrifuge cells at about 850 rpm for 3-5’ (push down lid all the way, then lock)
7) aspirate supernatant, leave about 1 mL to avoid aspirating the pellet
8) resuspend pellet in 5 mL of medium w/ serum and pipet up and down one time gently
9) add 5 mL more of medium w/ serum
10) transfer to T75 tissue culture flask, swirl gently, indicate cell line, passage number, and date
11) place tissue culture flask in incubator (37 C, 5.0% CO2), unscrew one turn on flask
12) observe following day
To split cells:
1) check cells under microscope to determine if ready to split (70-80% confluence)
2) warm-up medium w/ FBS (10%) at 37C
3) aspirate supernatant
4) wash cells with 5 mL PBS
5) add 2.5 mL trypsin/EDTA (stored @4C)
6) incubate at 37C 10+’
7) check under microscope if cells are dislodged
8) add 5 mL med w/ FBS
6) transfer into 15 mL tube
7) spin at 850-900 rpm 5’ to pellet
8) aspirate sup
9) resuspend pellet in 5 mL med w/ FBS
10) add~300 µL into 10 mL of med w/ FBS
11) count cell concentration under scope with hemocytometer
(# counted per corner 4x4 large squares)(104) cells per mL of medium
To subculture cells:
1) place medium in 37C bath
2) aspirate medium (serum inactivates trypsin)
3) wash with PBS 5.0 mL
4) aspirate PBS
5) add 1.5 mL of Trypsin 0.25% and swirl
6) incubate 3-5’ at 37C
7) examine under microscope (are they detached from the surface?)
8) add 5 mL medium w/ 10% FBS
9) transfer to 15 mL centrifuge tube
10) centrifuge at 850-900 rpm 5’
11) aspirate medium from centrifuge tube (leave pellet)
12) add 5 mL of medium w/ 10% FBS - measure cell density
1
13)
14)
15)
16)
resuspend cells
to new flask* add 9.5 mL of fresh medium w/ 10% FBS
transfer 0.5 mL of resuspended cells to new flask (1:10 dilution of cells)
label and date flask and indicate passage number
*may use same flask if one rinses w/ PBS, but greater chance of contamination
Procedure from ATCC: remove medium, rinse with fresh 0.25% trypsin solution, remove trypsin
and let the culture sit at room temperature (or at 37C) until the cells detach (about 10 minutes).
Add fresh medium, aspirate and dispense into new flasks. Subculture every 6 to 8 days
To transfect cells w/ LF2000:
1) on day before transfecting, place approx. 1.5 x 105 cells per well of a 24-well plate
2) dilute 1 µg DNA + 50 µL medium w/o serum
3) dilute 2 µL LF2000 + 50 µL medium w/o serum – leave at room temp 5’
4) mix DNA and LF2000 dilutes – leave at room temp 20’
5) add mixture to wells and swirl to mix
6) at 37C O/N
To harvest cells and isolate RNA:
1) aspirate supernatant from wells
2) for 24-well plate, add ~250 µL TRIzol reagent (1 mL per 10 cm2)
3) tilt plate and pipet up and down
4) place mixture into eppendorf tube
5) at room temp for 5’
6) add 50 µL chloroform
7) shake and mix
8) at room temp 2-3’
9) spin at 10,000 rpm 15’ @4C
10) aspirate upper layer into new tube
11) add 125 µL isopropanol (approx. 0.5x amount TRIzol added)
12) at room temp for 10’
13) spin at 10,000 rpm 10’ @4C – will see visible pellet
14) remove sup
15) add 250 µL 75% EtOH (approx. 1 mL 75% EtOH per 1 mL TRIzol added) (or cold 100%)
16) spin at 14,000 rpm 5’ at room temp
17) remove EtOH
18) within 5-10’, add 20 µL ddH2O
19) store at -80C
To reverse transcribe total RNA:
1) make oligodT mix –
0.25 µL
100 µM oligodT
5.3 µL
ddH2O
5.55 µL
total each reaction
2) add 0.85 µL total RNA to 5.55 µL oligodT mix
3) at 70C 5’
2
4) on ice to cool
5) spin briefly to collect mix at bottom
6) make AMV mix 0.4 µL
AMV reverse transcriptase
2 µL
5x AMV buffer
1 µL
10 mM 10x dNTP
0.2 µL
Ribonuclease III Inhibitor/RNasin
3.6 µL
total each RT reaction
7) add 3.6 µL AMV mix to total RNA with oligodT mix
8) at 37ºC for about 1 hour
To rtPCR cDNA:
1) make reaction mix -
1 µL
10x Buffer B
1 µL
2 mM dNTP
0.6 µL
25 mM MgCl2
0.3 µL
50 µM reverse primer
0.3 µl
50 µM forward primer
0.075 µL
Taq polymerase
5.725 µL
ddH2O
9 µL
total each rtPCR reaction
2) add 1 µL RT product to 9 µL rtPCR mix on ice
3) run PCR program
To freeze cells:
1) check cells under microscope to determine if ready to split (70-80% confluence)
2) warm-up medium w/ FBS (10%) at 37C
3) aspirate supernatant
4) wash cells with 5 mL PBS
5) add 2.5 mL trypsin/EDTA (stored @4C)
6) incubate at 37C 10+’
7) check under microscope if cells are dislodged
8) add 5 mL med w/ or w/o FBS
6) transfer into 15 mL tube
7) spin at 850-900 rpm 5’ to pellet
8) aspirate sup
9) resuspend pellet in 5 mL med w/ FBS
10) add ~300 µL into 10 mL of med w/ FBS
11) count cell concentration under scope with hemocytometer
(# counted per corner 4x4 large squares)(104) cells per mL of medium
12) resuspend cells in freeze medium for conc of about 2x106 cells/mL
13) add 1 mL to cryovial
14) in ice for 60’
15) at -20C for 60’
16) at -80C O/N
17) transfer to liquid nitrogen
3