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Preparation of Lymphocyte Cell Suspension

Introduction

The mouse spleen will serve as a source of lymphocytes to be fused with mouse myeloma cells to form hybridoma cells. The spleen is the source used most often as it is easily obtainable. Lymph nodes and peripheral blood are also occasionally used as lymphocyte sources. In humans, peripheral blood is often the only source used, unless the spleen or some lymph nodes were being surgically removed (and thus available).

Procedures

a. Spleen removal (you are not required to sacrifice the mouse - if you prefer, the instructor will sacrifice it for you)

1. Set up a sterile glass petri dish containing about 5-10 mls of cold, sterile Hank's Balanced Salt Solution (HBSS) before starting to remove the spleen from the mouse. You will place the spleen into this petri dish, and it is much easier to have it set up ahead of time.

2. Remove a mouse from the cage by grasping its tail with your finger tips. Place it on the wire cage cover and allow it to pull forward while still grasping its tail.

3. Place a pencil at the base of the neck and firmly push down on it to hold the animal in place and to prevent the animal from pulling its head away.

4. This next step takes a little getting used to. Pull on the tail to cause cervical separation (and immediate death). You may feel a "crunching" effect as the head separates from the spinal cord). The animal may "twitch" for a few seconds, but will become immobile very quickly.

5. Place the dead mouse on its back or right side on a clean surface. Wipe the abdomen and left side thoroughly with alcohol to clean it off and to wet the fur - this is not going to sterilize the animal's surface, but as long as you don't let the spleen touch the surface, no contamination will result.

6. Wearing sterile gloves (if desired) and using one set of sterile forceps and scissors, carefully trim back the fur and skin.

7. Using the second set of sterile forceps and scissors, cut a slit into the abdomen. Enlarge the slit with the scissors.

8. Using the same (second) set of sterile forceps, locate the spleen by gently pushing the stomach to the animal's right side. The spleen will appear as a dark red, thin, flat organ about one inch in length by one-quarter inch wide.

9. Hold the spleen with the sterile forceps and cut it free using the sterile scissors.

10. Place the spleen in a sterile, glass petri dish containing a few mls of cold, sterile HBSS. Trim away and dispose of excess fatty or connective tissue.

b. Spleen cell dissociation

1. Unscrew the cap from a 15 ml sterile centrifuge tube, but leave the cap setting on the top of the tube, with the tube upright in a wire tube rack. Do NOT touch any portion of the top of the centrifuge tube, as you want it to remain sterile.

2. Carefully unwrap a foil-wrapped, sterile filter unit to which has been attached a fine-gauge needle.

3. Make sure the needle is firmly attached to the end of the filter unit

You may want to use the edge of the needle cover to push on the shoulder of the needle to firmly anchor it to the filter unit. BE CAREFUL when removing the needle cover so you do not puncture yourself with the needle. You also want to be sure you do not remove the needle from the filter unit as you remove the needle cover. If the cover is "stuck" on the needle, ask the instructor to help you remove it properly. Replace the cover on the needle after using the cover to anchor the needle to the filter unit.

4. Unscrew the filter unit. Make sure the black, rubber o-ring remains in the top of the unit (the portion without the needle attached). If the O-ring remains stuck to the bottom portion of the unit, you may need to gently touch the O-ring with the top portion of the filter unit to dislodge it and have it fall into the inside of the top portion.

5. Place the top portion of the filter unit (with rubber O-ring inside) aside on a sterile surface - the inside of the foil wrap is a good location.

6. CAREFULLY remove the needle cover from the needle without dislodging the needle from the filter unit. If the cover is "stuck" on the needle, ask the instructor to help you remove it properly. You can set the needle cover aside anywhere as you will no longer need it. However, keep it to cover the needle when you discard it into a special container.

7. Remove the loose cap from the centrifuge tube and set the cap down on a sterile surface (e.g., the inside of the foil wrap).

8. Place the bottom portion of the filter unit (with needle attached) on top of the open centrifuge tube. The filter unit will set on top of the tube and the needle will extend down into the tube.

9. Using sterile forceps, place the spleen onto the fine wire mesh in the bottom part of the filter unit.

10. Add a very few drops of cold, sterile HBSS to the spleen on the wire mesh to keep it moist. Take care not to add too much salt solution to prevent spillage over the edge of the filter unit.

11. Open a sterile 5cc syringe. Remove the plunger but do not set it down anywhere. Place the barrel of the syringe on a sterile surface (e.g., inside of the foil wrap).

12. Press firmly on the spleen with the rubber end of the plunger portion of the syringe to force the spleen to break apart and the cells to pass through the wire mesh. Gentle "grinding" of the spleen across the mesh with the plunger will help to hasten the breaking apart of the tissue. You may have to add a few drops of balanced salt solution to keep the tissue/cells moist. Be careful not to let the cells in suspension spill over the edge of the filter unit.

13. After you have completely dissociated the spleen cells (only the connective tissue and fatty tissue will remain behind on the wire mesh - it usually takes only 1-2 minutes to dissociate the spleen cells), firmly screw on the upper portion of the filter unit (be sure the rubber gasket is in place).

14. Using sterile forceps, remove and discard the plug that is in the small opening of the upper tip of the top portion of the filter unit.

15. Screw the tip of the syringe barrel onto the upper portion of the filter unit. Using a sterile pipette, add 5 ml of sterile balanced salt solution to the syringe barrel. Place the rubber-tipped syringe plunger into the barrel. Gently push the salt solution through the wire mesh with the syringe plunger to wash the dissociated cells into the centrifuge tube. The needle will break apart any small clumps remaining after passing the dissociated spleen through the wire mesh.

16. Unscrew the syringe from the filter unit. Remove the plunger and set in on a sterile surface. Replace the barrel onto the filter unit. Using a sterile pipette, add 5 more mls of sterile balanced salt solution to the syringe barrel, insert the plunger and push the salt solution through the wire mesh. You will have about 10 mls of a well dissociated cell suspension in the centrifuge tube when you finish.

17. Remove the filter unit and needle from the centrifuge tube. You are now finished with it so you can set it down anywhere (do not forget to eventually remove the needle and discard it properly. Also, you will eventually need to unscrew the filer unit and wash the screen before returning the filter unit parts).

18. Screw the cap back onto the tube and gently centrifuge the cell suspension (200 x g) for 3-4 minutes. If you are using an IEC centrifuge, a setting of 3 will give you the proper centrifuge speed and "g" force.

19. Discard the supernate. If you centrifuged the suspension properly, you can carefully pour it off and the cell pellet will remain in the bottom of the tube.

20. Add 5 ml of cold, sterile HBSS and resuspend the cells using a sterile pasteur pipette and sterile pipette bulb.

c. Cell count and cell viability

Using a hemocytometer (with grid lines), sterile 0.4% trypan blue, and Hank's Balanced Salt Solution (as a diluent), estimate the total number of viable lymphocytes/ml of the suspension as follows:

1. Using separate, sterile pipettes for each addition, mix 0.1ml of the well-resuspended cell suspension + 0.1ml of 0.4% trypan blue + 0.8ml of sterile HBSS in a small, glass tube. The glass tube need not be sterile, as you will not be using the cells that are being counted for any purpose other than to determine the cell concentration of your cell suspension.

2. Allow this mixture to stand at room temperature for about 1 minute. Do NOT let stand for much longer than one minute before counting. Cells will begin to die if held too long in the trypan blue mixture, and you will then have an inaccurate count of cell viability.

3. Using a Pasteur pipette (need not be sterile), carefully fill one chamber of the hemocytometer under the coverslip. Using a light microscope, count the number of viable lymphocytes in the area of the hemocytometer you select (it is suggested that you count cells in all four large corner squares). Your lab instructor will assist you to identify lymphocytes if you do not already know what they look like (two lymphocytes in this image - dark blue-stained nucleus fills almost the entire cell). There will be other cells types in the spleen cell suspension that you do not want to count, e.g., red blood cells (most of the light reddish-brown round cells in this picture), monocytes (the nucleated cell at the right in this image is a monocyte, monocytes are usually larger than lymphocytes), polymorphonuclear neutrophils (PMNs) (the two nucleated cells at the right edge of this image are PMNs).

You will have to be patient to count the cells as they may be in a high concentration compared to most cell suspensions you may have counted previously (diluting the stained cell suspension with HBSS should give a reasonable cell concentration that will allow you to recognize and accurately count the lymphocytes present).

4. Calculate the number of viable lymphocytes/ml using the following equation:

If you counted 4 large squares, then the # of squares counted in the denominator would be 4. If you counted more or less than four large squares, adjust the denominator accordingly. If you counted small squares in any of the large, corner squares, each smaller square is 1/16th of a large square. You will need to adjust for this counting of partial large squares. For example, if you counted a total of only 6 smaller squares in one of the large, corner squares, then the denominator in the above equation would be 6/16. Make sure you know how many small squares are in any large square you may be using (if you decide to count cells in small squares, instead of large squares). Some of the large squares have 20 small squares or 25 small squares (and each of these small squares would then be 1/20 or 1/25 of a large square).

5. Determine the volume of this lymphocyte suspension you would need to perform a fusion.