Brad St. Croix, December, 1998
EC isolation procedure usually takes ~13 -14 hours. Leaving intact specimens O/N is possible but results in ~10-fold
less yield. Yields
usually range from ~2 x105
to 1 x 106 cells. Although tumor EC yields are typically higher than EC yields
from normal mucosa, yields from the latter are highest
when samples are taken from thick muscosa, especially
from the ascending colon.
At pathology lab, strips (~0.5 cm x 10 cm)
containing mucosa and submucosa are removed from a normal
section of colon with scissors by cutting just overtop
of the underlying muscle.
Place strips immediately on ice in a 50 ml tube
containing DMEM+ (see Stocks).
A 50 ml tube at least ¾ full (~35 ml)
is usually sufficient for normal.
The colon tumor is carefully resected with a
razor blade and also placed in DMEM+ on ice. Half of
a large tumor (~golf ball size) is usually sufficient.
Before beginning, if necessary, tiny sections
of tissue are removed, rinsed in PBS, and frozen in
OCT compound in a dry ice/ methanol bath (or fixed in
4% paraformaldelyde and then frozen) and stored at -80°C for later
First, crypts are removed from the normal mucosa
while the tumor sample is stored on ice.
This step removes enterocytes which bind non-specifically
to Dynabeads especially as they die.
Rinse mucosa with cold (4°C) PBS, pat
with Kimwipe to remove mucous and blood, rinse again,
pat again, then place in PBS in 50 ml tube on ice.
Weigh boats are convenient for rinses.
Place rinsed mucosa strips into 5 mM DTT in
PBS (RT). I
usually split a ¾ full tube of mucosa into two 50 ml
tubes each containing 20 ml of DTT.
Leave at RT for 20 min.
Transfer mucosa strips into 10 mM EDTA in PBS
(4°C) and leave
on ice for ½ hour.
I usually use four 50 ml tubes each with 20 ml
of EDTA solution for each ¾ full tube of starting material.
After ½ hour, transfer strips to new tubes with
fresh EDTA solution and incubate a further ½ hour on
ice (i.e., one hour total).
During EDTA incubation prepare fresh collagenase
A digestion cocktail.
I usually prepare 160 ml of 2 mg/ml Collagenase
A, 250 ug/ml elastase, 25 ug/ml DNAse I (all from Boehringer
Mannheim) in DMEM+.
The diluted collagenase and elastase should be
filtered before using.
The 160 ml solution is split into eight 50ml
tubes each containing 20 ml and left on ice until ready
After EDTA incubation, transfer cells to new
50 ml tubes containing 30 - 40 ml cold (4°C) PBS. Shake back and forth 10 - 20 times.
Individual strips are placed crypt-side-up on
a weigh boat, and crypts are scraped off using 2 glass
stripped lamina propria and submucosa are collected
in PBS on ice.
The lamina propria and submucosa are minced
into small pieces with 2 razor blades on a large weigh
boat. Cut pieces are usually split into four 50ml tubes
(from a ¾-full tube of starting material) each containing
20 ml collagenase A digestion cocktail.
Likewise, the minced pieces from half of a golf-ball-size
tumor are also collected into four tubes each containing
20 ml of collagenase A cocktail.
in collagenase mix are incubated in a bacterial shaker
on a diagonal 50 ml tube rack for 1-1/2 to 2 hours at
37°C, 120 rpm.
I found tissue disruption to be significantly
enhanced by pipetting each sample 10-15 times back and
forth with a 25 ml pipette every 20-30 minutes.
After 1-1/2 hours the tumor digest is filtered
sequentially through 500 um, 250 um, 100 um and then
40 um nylon filter mesh (Tetko).
Next, the normal sample is filtered through 500
um, 250 um, 100 um and then 40 um.
Filtered samples are split into several 50
ml tubes and volume is increased to 40 ml with
cold PBS/BSA (cells and media should be kept on ice
or at 4oC from this point on).
After pelleting (1200 rpm, 15-20 min,
cells are rinsed with 40 ml of cold PBS/BSA.
Some clumping can occur at this stage, which
may be related to the amount of extracellular particulate
matter present. Clumping appears to be minimized if several tubes are used
during the rinses.
I usually use twelve 50ml tubes (4 for normal
and 8 for tumor).
Repeat rinse (at least once) until solution above
pellet appears clear and resuspend pellets in volume
necessary for Percoll (see below).
Before adding to percoll gradient, cells are
re-filtered through 40 um mesh to remove spontaneously
Cells are separated based on density through
a preformed 30% Percoll gradient.
This step removes platelets and RBCs which can
cause clumping of the magnetic beads.
Gradients are prepared ahead of time in 15 ml
ultracentrifuge tubes (see stocks).
If too many cells are loaded onto a single gradient,
cells will stick to each other and migrate as a single
I typically use 8 tubes for cells from the normal mucosa,
and at least 28 for cells from the tumor.
Cells are resuspended in 4 ml (normal) or 14
ml (tumor) PBS/BSA, and 0.5 ml is slowly dispensed with
a Pipetman onto the side of the tube just above the
Cells are separated at 800 x g for 15 minutes
In the cold room, the top 5 ml
of each tube of Percoll (which contains the majority
of endothelial cells) is carefully removed with a pipetteman
and placed into a 50ml tube containing ~40 ml of PBS/BSA,
thus diluting the Percoll.
are pelleted (1200 rpm, 15 minutes), resuspended in
1ml PBS, and transferred through 25 um nylon filter
mesh (Tetko) into a 2 ml microfuge tube. Cells are pelleted
in a microcentrifuge (7 min; 600 x g; 4°C).
The remaining enterocytes and tumor cells which
can bind non-specifically to beads in the final magnetic
separation are removed using M450 beads which are prebound
to the BerEP4 antibody against epithelial specific antigens
(Dynal cat # 112.07).
Likewise, most of the remaining leukocytes are
removed using a cocktail of anti-CD45 (leukocyte common
antigen) beads (Dynal cat # 111.19), anti-CD14 beads
(Dynal cat # 111.11) and anti-CD64 beads (see appendix).
Both enterocytes and hematopoietic cells can be removed
simultaneously by pre-mixing their respective beads
the protocol below describes their sequential removal
which allows the recovery of independent epithelial
and hematopoietic cell controls.
The incubation with these beads should be kept
short, as endothelial cells can also bind non-specifically
After prerinsing BerEP4 beads 2 - 3x in PBS/BSA,
beads are added to cells and rotated at 4°C, 10 minutes.
Typically, I resuspend the cells in 500 ul (normal)
or 1000 ul (tumor) of PBS/BSA.
Then, I add 50ul (normal) or 250ul (tumor) of
Regardless of the cell numbers I would not recommend
resuspending the cells in less than 500 ul or using
less than 50 ul of beads.
After an 8 min rotation at 4oC,
the bead/cell mix is immediately transferred to a 50
ml tube and diluted by raising the volume to 3 ml (normal)
or 15 ml (tumor) with PBS/BSA.
To remove enterocytes, the diluted bead/cell
mix is then transferred back into 2 ml microfuge tubes
(~1.5 ml/tube) and placed on a magnet for 2 minutes.
The supernatent (containing endothelial cells and hematopoietic
cells) is transferred to 2 (normal) or 10 (tumor) microfuge
tubes, pelleted (7 min 600 x g), and combined into a
single 2 ml tube containing 500 ul (normal) or 800 ul
(tumor) PBS/BSA. The left over bead bound enterocytes can be resuspended in
a small volume of PBS/BSA and stored on ice.
of hematopoietic cells.
Beads recognizing hematopoietic antigens (i.e.
anti-CD14, anti-CD45 and anti-CD64 beads) are premixed
together in a 1:1:1 ratio.
After rinsing the beads 3X and resuspending them
in their original volume (i.e. total volume after premixing),
220 ul (tumor) or 30 ul (normal) of beads are added
to the cells.
Following a 5 min rotation at 40C,
the bead/cell mix is immediately transferred to a 50
ml tube and diluted by raising the volume to 3 ml (normal)
or 15ml (tumor) with PBS/BSA.
Again, the diluted bead/cell mix is transferred
back into 2ml microfuge tubes (~1.5ml/tube) and placed
on a magnet. The
supernatent is collected into new microfuge tubes and
the magnetic separation is repeated once to ensure the complete removal of all beads-bound
the supernatent (containing endothelial cells) is transferred
to 2 (normal) or 10 (tumor) microfuge tubes and pelleted.
After pelleting, I usually resuspend cells in
300 ul (normal) or 800 ul (tumor) PBS/BSA. The left
over bead bound hematopoietic cells can be resuspended
in a small volume of PBS/BSA and stored on ice.
Cells are stained for 1 hour on ice with a
1/100 dilution P1H12 anti-endothelial cell antibody
(Chemicon cat# MAB16985).
Meanwhile, beads linked to secondary antibody
(Dynal M450 goat anti-mouse cat # 110.05) are washed
2-3x in PBS/BSA.
After rinsing cells 2 -3
times, prewashed beads are added to cells and
rotated at 4°C 8 minutes.
Typically, I use 50 ul (normal) and 100 ul (tumor)
of beads and resuspend the final cell/bead mixture in
a total of 800 ul (normal) and 1200 ul (tumor) of PBS/BSA.
Immediately following the incubation, PBS/BSA
is added to the samples to bring up the volume and samples
are usually spit into several tubes (e.g. 2 tubes normal;
4 tubes tumor).
Cells are placed on a magnet for 2 minutes. Bead-bound cells are resuspended
The magnetic separation is repeated until no
bead-free cells can be observed under the microscope
(usually 7 - 8 X).
After the final rinse, I usually resuspend
endothelial cells in 225 ul (normal) and 425 ul (tumor)
of PBS. To
count cells, 5 ul is removed from each sample (including
enterocyte and hematopoietic controls) and mixed with
5 ul of Berts magic solution containing DAPI.
Fluorescent nuclei are counted with a hemocytometer
using the DAPI filter. As well, 20 ul is removed from each sample to check EC purity
by immunofluorescent staining (see staining protocol). The remaining sample is placed on a for 2 minutes and usually
resuspended in 100 ul of lysis/binding buffer (eg. Dynal
mRNA isolation kit) for each 100,000 cells.
After pipetting several times, beads are removed
from the sample and the lysate is stored frozen at -80°C.
DMEM + =DMEM + 1% FCS + 20 mM HEPES + P/S
100 mM DTT in PBS frozen as 1 ml aliquots
50 mM EDTA (pH 7.4)
Collagenase A- weigh out fresh
Elastase- 10mg/ml in PBS frozen as 4 ml aliquots
DNAse- in PBS frozen as 1 ml aliquots
PBS/BSA- PBS + 0.5%BSA
Preformed Percoll gradients
1st make up stocks:
1) 10 x PBS (store RT)
2) dilution buffer: 1x PBS + 7.5 mM HEPES + 0.5%
BSA + 1x P/S
Under a sterile hood, 90 ml of Percoll is mixed with
10 ml of 10 x PBS. A 30% Percoll gradient is made by
mixing 3 ml of PBS buffered Percoll with 7 ml of dilution
buffer (final pH = 7.4) in Beckman ultra-clear centrifuge
tubes (Cat #344085).
Tubes are spun at 17,000rpm or 20,000x g for
20 minutes (20°C; acceleration
4; deceleration 4) using the 50Ti rotor in the digital
ultracentrifuge (or 17 min in the non-digital ultracentrifuge).
Preformed gradients can be stored at 4°C for several days if kept air tight.
staining of human colon sections reveals intense staining
for CD64 in the large majority of non-epithelial cells,
most of which appear to be macrophages.
Antibodies against CD14 and CD45 also stain most,
if not all, hematopoietic cells, albeit with less intensity.
Importantly, no cross reactivity of any of these
antibodies with endothelial cells (as determined by
double staining with P1H12 anti-endothelial cell antibody)
is evident. High CD64 expression levels make this surface marker particularly
valuable for removing hematopoietic cells.
However, Dynal does not yet sell beads pre-conjugated
to anti-CD64 antibodies, although they do for CD14 and
CD45. Nevertheless, such beads can be prepared by mixing CD64 antibodies
with secondary antibody-linked dynabeads as follows:
anti-CD64 antibody-linked dynabeads.
Remove 1ml of goat anti-mouse IgG secondary linked-beads
(Dyanl cat # 110.05) and place in a microfuge tube.
This represents 30mg of beads.
Using a magnet rinse beads twice with PBS/O.1% BSA.
Resuspend beads in 600 ul to obtain a 50mg/ml concentration
Add 60ul of mouse anti-human CD64 antibody (Pharmingen
cat # 31841A) (since the antibody stock is 0.5ug/ul
this represents ~1ug antibody/ mg of beads).
Rotate at 4oC
Remove supernatent, add 1ml PBS/O.1% BSA and rotate
5 min at 4oC.
Repeat step #5 four times.
in 1ml of PBS/O.1% BSA + 0.02% sodium azide and store
at 40C until ready
to use. The
beads can be stored for several weeks but should be
rinsed to remove the azide before using.
Immunofluorescent Staining of Human Endothelial Cells
resuspend bead-bound cells (already labeled with
P1H12 anti-endothelial cell antibody) with a 1/50 dilution
of Alexa anti-mouse secondary antibody diluted in PBS
containing 0.5% BSA (PBS/BSA).
Incubate 20 min at 4°C.
rinse 3X with PBS/BSA
resuspend in 10-20 ul of DABCO antifade solution
and place on slide.
that vWF is located in intracellular Weibel-Palade bodies
and is not a membrane protein, so you have to permeabilize
and fix cells.
resuspend bead-bound cells in 50 ul PBS/BSA.
To fix cells add 100 ul Reagent A from Leucoperm
fixation kit (Serotec cat #BUF09), leave 15 min at RT.
add 1ml PBS/BSA, spin (600 x g, 7 min), and rinse
1X with PBS/BSA. (The samples can be stored at this
stage for at least 2 days without significant loss of
cells or staining.
If storing, rinse 1X more and leave in PBS/BSA
on rotator in cold room)
Add 200 ul undiluted goat serum, leave 15 min at
resuspend pellet in permeabilization/antibody cocktail
ul Reagent B (Reagent B is the permeabilization reagent
with the Leucoperm fixation kit (serotec cat
ul goat serum
ul rabbit anti-human von willebrand factor antibody
(Dako) (final 1/400 dil.)
ul mouse anti-human EC antibody (clone P1H12, Chemicon
cat# MAB16985 (final 1/100 dil.)
incubate 30 min at RT.
rinse 1 X with PBS/BSA
add cocktail of secondary antibodies in 100% goat
incubate 20 min RT.
1X with PBS/BSA.
1/500 dilution of rhodamine-linked streptavadin in 100%
20 min RT.
1 ml of DAPI
(0.1 - 0.3 ug/ml).
Incubate 3 min.
2 - 3X with PBS/BSA
in 10-20 ul of DABCO antifade solution (Sigma cat # D-2522),
and place on slide.