Brad St. Croix, December, 1998

 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 applications. 

 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 to use.

 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 slides.  The 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.  

 Tissues 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, 4°C), 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 forming clumps.

 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 blob.  Therefore, 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 Percoll.  Cells are separated at 800 x g for 15 minutes (4°C).

 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.  Cells 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 together.  However, 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 to beads.

 Removal of enterocytes.  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 BerEP4 beads.  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 4^oC, 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. 

 Removal 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 cells.  Finally, 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  in PBS/BSA.  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 Bert’s 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.

Stock solutions

  “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

       (store at 4°C)

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.

Appendix

Immunofluorescent 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:

Preparing anti-CD64 antibody-linked dynabeads.

1.      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.

2.      Using a magnet rinse beads twice with PBS/O.1% BSA. 

3.      Resuspend beads in 600 ul to obtain a 50mg/ml concentration of beads. 

4.      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 4^oC 1 hour.

5.      Remove supernatent, add 1ml PBS/O.1% BSA and rotate 5 min at 4^oC.

6.      Repeat step #5 four times.

7.        Resuspend 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.

Single stain

8.      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.

9.      rinse 3X with PBS/BSA

10. resuspend in 10-20 ul of DABCO antifade solution and place on slide.

Double stain

Note that vWF is located in intracellular Weibel-Palade bodies and is not a membrane protein, so you have to permeabilize and fix cells.

1.      resuspend bead-bound cells in 50 ul PBS/BSA.

2.      To fix cells add 100 ul Reagent A from Leucoperm fixation kit (Serotec cat #BUF09), leave 15 min at RT.

3.      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)

4.      Add 200 ul undiluted goat serum, leave 15 min at RT.  Pellet, remove supernatant.

5.      resuspend pellet in permeabilization/antibody cocktail containing:

200 ul Reagent B (Reagent B is the permeabilization reagent that comes             with the Leucoperm fixation kit (serotec cat #BUF09)

17.3 ul goat serum

0.5 ul rabbit anti-human von willebrand factor antibody (Dako) (final 1/400 dil.)

2.2 ul mouse anti-human EC antibody (clone P1H12, Chemicon cat# MAB16985 (final 1/100 dil.)

6.      incubate 30 min at RT.

7.      rinse 1 X with PBS/BSA

8.      add cocktail of secondary antibodies in 100% goat serum:

1/50 alexa anti-mouse

1/50 biotin-anti-rabbit

9.      incubate 20 min RT.

10.  rinse 1X with PBS/BSA.

11.  add 1/500 dilution of rhodamine-linked streptavadin in 100% goat serum.

12.  incubate 20 min RT.

13.  pellet cells.  Add 1 ml of  DAPI (0.1 - 0.3 ug/ml).  Incubate 3 min.

14.  rinse 2 - 3X with PBS/BSA