1. Removal of the Spleen
1) Kill the mouse by cervical dislocation.
2) Lay mouse on dissecting board, “left side” uppermost.
3) Surface-sterilize the skin using 70% ethanol or a proprietary compound.
4) Using one set of sterile surgical instruments (forceps and scissors), cut through the skin just below the ribcage and visualize the spleen.
5) Using a second, smaller, set of instruments, remove the spleen, trimming away any fatty tissue.
6) Place spleen into complete medium at room temperature (see Note 10 ). Spleens from multiple animals can be pooled.
2. Preparation of a Single-Cell Suspension using a Metal Sieve
This has been our routine method for many years. It avoids the use of pro-teolytic enzymes, gives good recovery of DC numbers, and, in conjunction with overnight culture and metrizamide separation, yields a mixture of CD8αα+ and CD8αα- DC (see Fig. 1 ).
1) Strain spleens by pouring through a sterile metal cell-strainer. Discard medium.
2) Place the strainer containing spleens into a 60 mm Petri dish and add a few milli-liters of fresh medium.
3) Using the barrel from a 2 mL syringe, press the spleens through the strainer. Continue until only a little fibrous tissue remains in the strainer.
4) Remove the strainer and place in upturned lid of the Petri dish.
5) Reinsert plunger into syringe barrel and use to transfer spleen cell suspension to a 10 mL conical tube. (A larger tube or replicate tubes will be required for multiple spleens.)
6) Using fresh medium and a Pasteur pipet rinse the Petri dish and the cell strainer to ensure that all cells have been recovered. Pool with the rest of the spleen cell suspension.
7) Top-up tube with complete medium to appropriate volume (see below).
3. Preparation of a Single-Cell Suspension using Collagenase
This method gives an overall increase in DC yield and improves the recovery of the tightly tissue-bound CD8αα+DC Fig. 1 ). The use of proteolytic enzymes may be undesirable for some applications.
1) Place 5 mL of digestion mix into a Petri dish.
2) Put a small needle (26G×1/2 in. is ideal) on a 1 mL syringe and fill with digestion mix.
3) Gently inject the first spleen with 0.5-1 mL of digestion mix. Initially insert the needle just inside the spleen at the narrowest part of the capsule. Inject approx 100 μL, advance the needle slightly, and then inject again. Continue in this fashion. This may take some practice. The spleen will distend and change from a dark maroon color to a reddish orange.
4) Using the needle tear open the spleen in a second (empty) Petri dish.
5) Place the spleen back in the first Petri dish containing the digestion mix.
6) Transfer the released cells in the second dish to a conical tube and place on ice. Rinse this dish with more digestion mix and pool with the other cells on ice.
7) Repeat steps 1-6 with the next spleen, pooling released cells and spleens.
8) Using a disposable scalpel or scalpel blade cut up spleens into small fragments.
9) Transfer to a T25 tissue culture flask and incubate with gentle shaking at 37°C for approx 60 min (see Note 11 ).
10) At the end of the incubation, collect contents of dish. Rinse dish with a small volume of digestion mix and pool together.
11) Press contents through a cell strainer as described previously.
12) Pool cells with those already on ice. Rinse dish in digestion mix or medium and again pool with the other cells.
13) Spin down (350g, 5 min), discard supernatant and gently resuspend cell pellet in 10 mL complete medium.
14) Spin down again and resuspend in complete medium. Adjust to required volume (see below).
4. Preparation of a Single-Cell Suspension by the “Manual Extraction” Method
This very gentle method yields DC that are almost exclusively CD8αα- Fig. 1 ), so this approach may be useful as an early step in purifying this subset.
1) Place some complete medium in a Petri dish and place the dish at an angle by resting it partially on its lid.
2) Using forceps, make a hole in the capsule at one end of the spleen.
3) Place the spleen on the sloping Petri dish, punctured end facing “down the hill.” Hold in place with forceps.
4) Using a cell scraper gently press cells out of the spleen by working the scraper from the middle to the lower end of the spleen.
5) When capsule is clear turn the spleen round, make a hole at the other end of it and work the rest of the cells out.
6) When all cells have been removed, discard the empty capsule and transfer the cells to a conical tube.
7) Repeat the procedure with additional spleens, pooling the released cells.
8) Adjust to the appropriate volume (see below).
5. Notes
1). 2-Mercaptoethanol is an inhibitor of collagenase. Therefore it should be omitted from medium used prior to the enzymatic digestion step if collagenase is used to produce a spleen cell suspension.
2). Collagenase D is recommended for maintenance of cell-surface protein integrity.
3). There can be considerable batch-to-batch variation in collagenase. Although a concentration of 1 mg/mL usually gives satisfactory results, it may prove necessary to adjust this concentration.
4). We use Falcon flasks. Products from other manufacturers may also be suitable, but there could be variation in performance. Small-tissue culture-grade Petri dishes may also be used.
5). Do not be tempted by the cheaper centrifugation grade metrizamide-it doesn’t work! We have no experience with metrizamide from other manufacturers.
6). It is important to avoid air bubbles in the buffer, as these will affect the performance of the separation column. Degassing of the buffer is recommended by the columns’ manufacturers. At the very least the buffer should be prepared and handled so as to mimimize frothing. To this end we prepare our buffer well in advance of use.
7). If in doubt about the sterility of monoclonal antibodies or immunomagnetic beads, small volumes can be sterilized by centrifugation through 0.22 μm Spin-X centrifuge tube filters (Costar; cat. no. 8160) at full speed in a microfuge for 2 min. When using beads, be sure to resuspend the pellet that forms in the bottom of the tube. All antibody concentrations stated are ones that we have found to work well in general, but investigators may need to vary these for their own applications.
8). “Fc-Block” is a mixture of unconjugated monoclonal antibodies to CD16 and CD32 (FcγIII/II), and, as its name suggests, it helps reduce nonspecific binding of the labeling antibodies via Fc receptors.
9). For best performance, precool the columns, magnets, and holders in a fridge or cold room. We also use “cool packs” supplied in the packaging of many cooled products, to keep the apparatus cool during prolonged separations.
10). If proceeding directly to preparation of cell suspensions from the spleen, it is better to keep the organ at room temperature than to expose it to the “shock” of the temperature changes involved in placing on ice and then warming up again in subsequent handling.
11). The length of the incubation may need to be varied with different batches of collagenase.
12). The performance of metrizamide gradients can be affected by small variations in osmolarity of the RPMI-1640 medium used to prepare them. The osmolarity of RPMI may vary slightly between manufacturers depending, for instance, on whether the medium is intended for use primarily with human or mouse cells. The osmolarity suitable for mouse cells is required for successful enrichment of DC. If your gradients perform poorly, it may be worth switching suppliers of RPMI.
13). There is anecdotal evidence that metrizamide gradients perform better when they have been through one freeze-thaw cycle. Therefore, we do not use freshly prepared metrizamide for separations.
14). Do not be tempted to remove tissue fragments from the cell suspension before overnight culture. These fragments are probably an important source of migrating DC.
15). Ideally use 10 mL of medium per spleen. This amount will then be cultured in two flasks and separated over two metrizamide columns. If processing many spleens, this can be reduced to 5 mL per spleen to reduce handling but do not be tempted to reduce this volume further. It is also inadvisable to “scale up” the separation procedure by using bigger flasks and columns.
16). The exact length of the “overnight” culture can influence the maturity and function of the DC obtained. Therefore, it is important to be consistent in the length of this incubation.
17). Do not allow the metrizamide to become too warm. This can be a problem in non-air-conditioned labs on hot summer days.
18). The pellet can be used as a source of lymphocytes. However, these cells need to be handled gently to allow them to recover from exposure to the hypertonic metrizamide.
19). Sudden changes in DC yields can be a sensitive indicator of the presence of infection within an animal facility.
20). The purity of DC separated on metrizamide varies with mouse strain. For instance, we routinely obtain better purity of DC from the spleen of BALB/c mice than from C3H mice.
21). To avoid frothing, resuspend gently using a pipet tip rather than by vortexing.
22). Incubation on ice is also possible but the time of incubation will need to be extended (20-30 min).
23). Wash thoroughly to ensure all EDTA is removed.
24). Flow through the column is much slower when the flow restrictor is in place. If flow stops, restart by gently pushing the plunger supplied into the syringe barrel. Push no more than is absolutely necessary to restart flow.
25). With the flow restrictor in place, the purity of the retained cells may be reduced. To improve purity of the retained CD45R+population, a pass over a second column with no restrictor in place may bring benefit.
26). The volume of beads added can be reduced to 50 μL without appreciable loss on recovery or purity of DC. Further reduction is not recommended.
27). When separating DC directly from whole spleen cells, this second pass over the column is essential for good purity. Expect up to 98% of the recovered cells to be MHC class II positive. Of these cells, up to 95% express CD11c. Expect to recover approx 2.5% of the starting spleen cell suspension.
