Stress Response Profiles in Human Fibroblasts Exposed to Heat Shock or Oxidative Stress - Protein Misfolding and Cellular Stress in Disease and Aging - page 159

3.1. Cell Culture
and Stress Treatments

1. Primary human skin fibroblasts are cultured in DMEM with
10% FCS (DMEM-10) at 37°C in humidified atmosphere of
5% CO 2 and passaged with trypsin-EDTA when approaching
confluence. Six 95% confluent 75 cm 2 culture flasks are
required for each stress treatment as well as for the “no stress
treatment”.
2. Heat shock treatment: Growth medium is changed on the day
before the stress treatment (see Note 2). Seal all culture flasks
and start the experiment by transferring cells to a 44°C heat
incubator. After 1 h at 44°C, culture flasks are unsealed and
moved back to the 37°C CO 2 -incubator for 24 h recovery.
3. Oxidative stress treatment: Prior to the stress treatment,
remove medium and rinse cells once in preheated (37°C)
PBS. Add 10 ml H 2 O 2 diluted in DMEM without serum
(600 µmol/L final concentration) to each culture flask and
incubate at 37°C (see Note 3). After 1 h, remove H 2 O 2 -
medium and rinse with preheated PBS. Then add preheated
DMEM-10 and move cells back to the 37°C CO 2 -incubator
for 24 h recovery.
4. “No stress treatment”: Cells remain in DMEM-10 at 37°C
for 24 h.
5. Rinse twice in PBS and scrape off cells from the six culture
flasks at different times after the stress treatment: 0, 2, 4, 8,
12, and 24 h. Cells from each culture flask are split in two
pellets (3 min at 1,500 rpm, 4°C) and stored at −80°C for
RNA- and protein analysis.

3.2. RNA isolation
and cDNA synthesis

1. Resuspend cell pellet corresponding to a half T75 culture
flask in 1.8 ml TRIzol TM Reagent, add 180 µl chloroform,
vortex 15 s, and incubate 5 min on ice (4°C).
2. Centrifuge at 12,000 × g for 5 min (4°C) and transfer the
H 2 O-phase (containing RNA) to a new 2 ml Eppendorf tube
(see Note 4).
3. Add another 540 µl chloroform, vortex, and centrifuge 5 min
at 12,000 × g (4°C). Transfer the H 2 O-phase to a new 1.5 ml
Eppendorf tube (see Note 4).
4. Precipitate RNA from the H 2 O-phase by adding an equal vol-
ume of isopropanol (ca. 450 µl), mix by hand, and incubate
at −20°C overnight.
5. Centrifuge at 15,000 × g for 15 min (4°C) and remove the
supernatant.
6. Carefully wash the RNA pellet in 180 µl 70% (v/v) ethanol,
centrifuge 1 min at 15,000 × g (4°C), and remove the
supernatant.
7. Resuspend the RNA pellet in 25 µl DEPC-H 2 O (mix by
pipetting). RNA can be stored at −80°C.