Dynamin cells
Maintenance Medium:
DMEM
(high glucose, +sodium pyruvate, Gibco # 41966-029)
10%
FBS
1%
PenStrep
400
µg/ml G418 (Geneticin, Gibco, now SIGMA,
50
mg/ml stock, top of the fridge
dilute
1:125, use sterile 1ml plastic pipette)
1
µg/ml tetracycline (Sigma)-> stock:2 mg/ml in freezer,
dilute
1:2000, use sterile filtertips
200
ng/ml puromycin (Sigma, P7255 or P8833) -> stock: 200 µg/ml
dilute
1:1000, use sterile filter tips
I use our normal HeLa medium (i.e. DMEM, 10% FBS, 1%
Pen/Strep, 1% MEM) and supplement the amount needed with G418, Puromycin and Tetracycline just before
use. Thaw a new aliquot of Puromycin and Tetracycline every two splits.
To split the cells, proceed as with Hela's. Do not
let the cells become too confluent. The cell bodies should not touch each other
at the time of splitting. In this case, the cells will easily disperse after
trypsinisation just by vigorous tapping against the side of the flask. If cells
are clumpy, titriate by pipetting up and down with a 10 ml pipette. Split the
cells 1:5 every two days.
Freezing medium:
DMEM
5%
DMSO
15%
FBS
10
mM Hepes pH 7.4
3
vials per 10 cm dish
Induction:
Wash
cells 3-5x with large volumes of PBS before trypsinising.
Split
cells and replate in medium without tetracycline.
Add
tetracycline to those dishes that require medium.
Split
1.5 x 10e6 cells per 10 cm dish.
Set
up one 6well dish with coverslips to monitor induction
by
immunofluorescence, use 1.5x10e6 cells/6well
Exchange
medium the next morning, to remove all traces of tetracycline
from induced cells
Grow
cells a total of 48 hours in the absence of tetracycline
Cells
ought to be no more than 75% confluent on the day of the experiment;
cells should not touch each other and there should be plenty of space between the cells!!!
To
check induction, incubate cells on one coverslip (-/+ Tet each)
for
15 min with biotinylated Transferrin at 25 µg/ml in DMEM.
Fix
all cells on coverslips with PFA as usual, permeabilise with 0.2% Triton-
X100 in PBS and stain either with HA/EEA1 followed by TexasRed/OregonGreen
or with HA alone (for those coverslips that were
exposed to Transferrin) followed by Streptavidin-OregonGreen.
Determine
% of cells expressing HA-K44A and % of cells taking Transferrin
up into the perinuclear recycling compartment.