Supplies: Ethanol 100% USP (highest quality) FACS Staining Buffer (1XPBS w/ 3% calf serum, 0.05% azide--filtered) —Dilute
staining antibodies in Buffer DNAse (Sigma D-5025, Bovine Pancreas) RNase (Boehringer, 25 mg bovine pancrease) Anti-BrdU-FITC (Becton Dickinson or Phoenix Flow) 0.15 M NaCl, 1.5 M NaCl 10% Paraformaldehype (kept as stock in -80°C) Tween 1M MgCl2
FACS Tubes
Protocol:
Cells in 96 well FACS plate
-
Block with 24G-2
-
Surface stain cells as usual -Omit fourth channel labeled antibodies on
all stains; EtOH
destroys APC
-
Prepare tubes from which to
transfer EtOH drop wise (1.2 ml EtOH on ICE)
-
Resuspend cells from 96 well
place with 100 µl 0.15M NaCl (cold)
-
Transfer to FACS tubes ON
ICE. Add 400 µl 0.15M NaCl to each tube
-
Vortex at 1/3 speed and add
EtOH with pasteur pipette at 1 drop per second. This is a critical step... do not add EtOH too quickly
-
Incubate on ice for 30 minutes
-
Spin 10 minutes @ 2000 RPM, 4° C
-
Dump and shake liquid into
waste
-
Using repeat pipetter,
squirt 1 ml FACS staining buffer into each tube
-
Spin 10 minutes and dump as
before (step 8)
-
Add 1 ml 1% paraformaldehyde
+ 0.05% Tween 10 -For 20 ml: 2.0 ml 10% paraformaldehyde 10 µl Tween-20
-
Incubate at room
temperature for 30 minutes
-
Incubate on ice for 30
minutes
-
Spin and dump as before
(step 8) Add 1 ml DNAse (0.15M NaCl + 4.2mM MgCl + 100 Kunitz units/ml DNAse)
-For 50 ml:
46.5
mL dH20 200 ul MgCl2 (1M stock) 1500 uL NaCl (5M stock) 100 Kunitz units Dnase (volume depends on activity of batch) Incubate for 30 minutes @ 25°
-
Spin 10 min. and dump as
before (step 8)
-
Transfer cells from FACS
tubes to 96-well plate. Wash once with staining media.
-
Block with 10% rat serum.
Incubate 15 minute on ice. Spin and dump as before (step 8: it is critical
to spin at high speed once the cells have been fixed with EtoH/ PFA since
they become less dense).
-
Add anti-BrdU-FITC or
biotin (1:20 dilution for Phoenix flow).
-
Pipette up and down to
resuspend pellet. Incubate for 30 minutes on ice (or overnight at 4°C).
-
Wash and dump as before.
Transfer cells into FACS tubes.
BrdU staining for cell cycle analysis (with HCl
treatment)
A total of 3x106
retinoblastoma cells were treated with cytokines, at the concentration noted
above, for 72 h in medium with 10% FBS. The cells were then pulsed with 20
μm BrdU (Sigma, St. Louis, MO) for one
hour at 37 °C in 10% FBS. The cells were then fixed with cold 95%
ethanol, washed with PBS and resuspended in 0.4 mg/ml pepsin in 0.1 N
HCl for 30 min at room temperature (RT) to release nuclei. The nuclei were
pelleted and incubated with 2 N HCl for 30 min at RT. Following
neutralization with 0.1 M Na2B4O7, nuclei were
pelleted and washed with PBS + 0.5% Tween-20 + 0.1% BSA. Nuclei were then
stained with anti-BrdU antibody (Becton-Dickinson, Franklin Lakes, NJ) for 90
min in the dark at RT. The nuclei were then washed with PBS, pelleted and
stained with FITC-labeled goat anti-mouse antibody (1:50, Becton-Dickinson) for
30 min at RT in the dark. The nuclei were washed with PBS, incubated with
propidium iodide (0.1 mg/ml) and RNAse A (10 μg/ml) overnight at 4 °C, and then
analyzed using a FACScalibur machine. Because of extensive aneuploidy, we chose
to quantitate only the non-aneuploid portions of the FACS. The portions of the
FACS analysis used for gating each population of cells were between 200 and 400
on the X-axis. The percentages, therefore, do not total 100%. However, the
percentages total to over 70% for all samples thus represent the majority of
cells in the population
.)cell starving:
24h with serumreduced Medium (5% serum)
.)Incubation with
e.g. TNF (24h)
for max.
stimulation of ICAM-1 we added IL-1ß (20 Units/ml) for the last 24h hours of
incubation
.)after incubation
wash cells with cold PBS, scrap the cells from the plate in
PBS+5%Serum into
a tube and put them on ice (no trypsin)
.)centrifugation:
4°C/1.500g/15min
.)cell pellet +
first antibody -we incubated the cells for 15 min on ice and shaked the
cells during this 15 min a few times (not vortexing)
.)stop the
reaction with 1ml PBS+5%Serum, centrifugation: 4°C/1.500g/5min
.)wash cells once
in PBS+5%Serum
.)cell pellet +
second antibody -we incubated 30 min on ice (sometimes shaking the cells)
.)stop the
reaction and wash cells as above, afterwards resuspend the cells in PBS for
FACS-analysis
The use of PBS
with 5% Serum may sound strange. We used this for a good treatment of the
sensitive primary endothelial cells. Could be that this is not necessary.
There is also a
publication which might be interesting:
KIM JA,
Atherioscler Thromb 14: 427-433, 1994
JC-1 (5, 5´, 6,
6´-tetrachloro-1, 1´, 3, 3´-tetraethylbenzimidazol-carbocyanine iodide) is a
lipophilic fluorescent cation that incorporates into the mitochondrial
membrane, where it can form aggregates due to the physiological membrane
potential of mitochondria. This aggregation changes the fluorescence properties
of JC-1 leading to a shift from green to orange fluorescence.
Intact living
cells stained with JC-1 therefore exhibit a pronounced orange fluorescence of
mitochondria, which is detectable by flow analysis (in the FL-2 channel).
Apoptosis results
in a break-down of the mitochondrial membrane potential and a subsequent
decrease of the orange fluorescence (and a slight increase of the green
fluorescence). By that means, apoptotic cells can be easily distinguished from
non-apoptotic cells.
JC-1 is prepared
as a 1000x stock solution in DMSO (5 mg/ml).
For the staining
of adherent cells it is diluted in medium to 5 µg/ml (with vortexing during the
dilution to prevent the formation of precipitates); the JC-1 containing medium
is added to the cells, followed by incubation for 10 min at 37°C (or RT for 15
min).
Subsequently the
cells are washed twice with PBS, trypsinized, suspended in 500 µl PBS and analyzed
by flow analysis.
Suspension cells
(lymphocytes): suspend 1:1 with 10 µg/ml JC-1 in medium (final conc.: 5 µg/ml)
Approximate
detection settings on FACSort:
FL1: 360 V (log)
FL2: 310 V (log)
Compensation :
FL1-7% FL2 und FL2-74% FL1
Andere Einstellungen:
Zelltyp-spezifisch, bei TF-1 z.B.:
SSC: 336 V lin
FSC: E00 lin 1.0
Threshold: FSC: 52
Example of an
analysis:
(modified from Andrea Cosarizza)
Adherent cells:
-
trypsinized
-
suspended in
medium + 10% FCS
-
centrifuged (1000
rpm, 5 min)
-
Pellet suspended
in PBS (1 ml)
Suspension cells:
Fixation with
EtOH:
Pipet cell
suspension into 2.5 ml absolute EtOH (final concentration approx. 70%)
(or vortex the
suspension at half speed while adding the EtOH) – to prevent clustering of
cells during the fixation. Incubate on ice for 15 min (or over night at –20°C).
Alternative
fixation with paraformaldehyde:
Pipet the 1 ml
cell suspension into 3 ml 4% paraformaldehyde and fix for 15 min at r.t.
Staining:
-
Pellet the cells
at 1500 rpm for 5 min
-
Suspend the
pellet in 500 µl PI-solution in PBS: 50 µg/ml PI from 50x stock solution (2.5 mg/ml) 0.1 mg/ml RNase A 0.05% Tritin X-100 Incubate for 40 min at 37°C
-
Add 3 ml of PBS,
pellet the cells (1500 rpm, 5 min) and take off the supernatant
-
Suspend the
pellet in 500 µl PBS for flow analysis (you can also leave about 500 µl of the diluted staining solution on the
pellet and suspend the cells in this solution > less loss of cells when
you take off the sup.) – the rest of the staining solution does not
interfere with the flow analysis.
Flow analysis:
Approximate
settings (on FACSort):
FL1: 570 V log.
(e.g. if you want to detect GFP):
FL2: 470 V
linear
Example:
|