Cell Cultures Splenocyte Cell Cultures

1. First, sacrifice the mice by CO2 asphyxiation. Working in a sterile hood, cut into the animal's skin below the front legs,and pull away the skin to expose the peritoneum and the underlying internal organs. Make a sterile incision into the peritoneal membrane on the animal's left side. Then remove the spleen with forceps. Pool together three spleens from CB2R+/+ and CB2R-/- mice groups and process them for primary splenocyte culture preparations.

2. Transfer the three spleens to a sterile 70-|im nylon mesh filter screen covering a 50-mL conical centrifuge tube containing 5 mL of complete media. Release the splenocytes from the spleens by gently massaging the spleens with the end of a 3-cc tuberculin syringe.

3. Carefully rinse the cell suspension in the filter screen with 15 mL of complete media to harvest all the spleen cells.

4. Centrifuge the cell suspension at 150g for 10 min at 15-18°C (CRU-500 Centrifuge, International Equipment Co., Neeham Heights, MA).

5. Discard the supernatant and add 5 mL of ACK lysing buffer to the cell pellet to lyse all red blood cells. Swirl the solution gently and place the tube on ice for 5 min with occasional swirling.

6. At the end of the 5 min, add 10 mL of complete media to the solution to stop cell lysis and centrifuge the cell suspension again at 150g for 10 min.

7. Discard the supernatant and add 20 mL of complete media to the cell pellet to remove any remaining lysis buffer. Gently resuspend the cells thoroughly.

8. To perform a cell count, a 1:20 dilution of the cell solution is needed. This is done by making a 1:10 dilution with complete media and then a 1:2 dilution with 0.4% stock concentration of trypan blue.

9. Count the cells using a hemacytometer. Then centrifuge the cell suspension at 150g for 5 min. Resuspend the cell pellet in 5 mL of serum-free media and dilute to 2 x 106 cells/mL in complete media.

10. For proliferation studies, plate the cells in complete media at 2 x 105 cells/0.1 mL/well into sterile flat-bottom, tissue-culture-grade 96-well microtiter plates. For cytokine studies, plate the cells in complete media at 2 x 106 cells/mL/well into sterile flat-bottom, tissue-culture-grade 24-well plates.

11. Treat specified wells with various final concentrations of the cannabinoid agonists (0,0.01 |M, 0.1 |M, 1 |M, 5 \xM). Using a repeater pipetor, treat the 24-well plates with 10-|L aliquots of the cannabinoid reagents (or 10 |L absolute EtOH as the non-cannabinoid-treated control), and treat the 96-well plates with 1 |L of the cannabinoid reagents (or 1 | L absolute EtOH as the non-cannabinoid-treated control). Set up the wells in the 24-well plates in triplicates. Set up the wells in the 96-well plates in quadruplicates.

12. After cannabinoid treatment, incubate the plates for 1 h in a humidified 5% CO2 incubator at 37°C. After 1 h of incubation, treat specified wells with the T-cell mitogen ConA at a final concentration of 2.5 |g/mL. Treat each well of the 24-well plates with 10 |L of ConA (or 10 |L RPMI as the non-ConA-treated control). And treat every well of the 96-well plates with 1 |L of ConA (or 1 |l RPMI as the non-ConA-treated control).

13. Incubate the plates at 37°C in a humidified 5% CO2 incubator for 72 h.

3.1.2. T-Cell Cultures

1. After obtaining the CB2R+/+ and CB2R-/- transfer the spleens to a sterile 70-|im nylon mesh filter screen covering a 50-mL conical centrifuge tube containing 5 mL of RPMI medium. Release the splenocytes from the spleens by gently pressing the spleens with the end of a 3 cc tuberculin syringe.

2. Carefully rinse the cell suspension in the filter screen with 15 mL of serum-free media to harvest all the spleen cells.

3. Perform a cell count using a hemacytometer by making a 1:10 dilution of the splenocyte suspension with serum-free media and then a 1:2 dilution with 0.4% stock concentration of trypan blue.

4. Transfer 5 x 108 total cells to a fresh 50-mL conical centrifuge tube.

5. Centrifuge the cell suspension at 300g for 10 min at 4-8°C.

6. Pipette off supernatant completely and resuspend cell pellet in 2 mL of column buffer.

7. Add 500 |L of biotin-antibody cocktail and incubate for 10 min at 4-8°C.

8. Add 1.5 mL of buffer and 1000 |L of anti-biotin microBeads and incubate for an additional 15 min at 4-8°C.

9. Wash cells with 10 mL of column buffer and centrifuge at 300g for 10 min.

10. Pipette off supernatant completely and resuspend cell pellet in 2500 |L of column buffer.

11. Place an LS column in the magnetic field of a suitable MACS Separator.

12. Prepare column by rinsing with 3 mL of column buffer.

13. Apply cell suspension onto the column. Allow the cells to pass through and collect effluent as fraction with unlabeled cells, representing the enriched T-cell fraction.

14. Wash the column with 12 mL of column buffer. Collect entire effluent in the same tube as effluent of previous step.

15. Perform a cell count by making a 1:10 dilution of the T-cell suspension with serumfree media and then a 1:2 dilution with 0.4% stock concentration of trypan blue.

16. Count the cells using a hemacytometer and determine the total number of T cells.

17. Centrifuge the cell suspension at 300g for 10 min at 4-8°C.

18. Resuspend pellet in a suitable volume of warm complete media to obtain a 2 x 106 cell/mL T-cell suspension.

19. Prepare a 5-10 |g/mL solution of anti-CD3 in sterile PBS.

20. Dispense 50 |L of the antibody solution in each well of the sterile flat-bottom, tissue-culture grade 96-well microtiter plates or 250 |L in each well of the sterile flat-bottom, tissue-culture-grade 24-well plates.

21. For the control unstimulated wells, add sterile PBS.

22. Tightly cover the plate with Parafilm to avoid sample evaporation and incubate at 4°C overnight.

23. Just before adding cells, remove the antibody solution with a multichannel pipet-tor.

24. Rinse each well two times with sterile PBS and discard PBS.

25. For proliferation studies, plate the cells in complete media at 2 x 105 cells/0.1 mL/well into sterile flat-bottom, tissue-culture-grade 96-well microtiter plates. For anti-CD3 stimulation, plate the cells in complete media at 2 x 105 cells/0.1 mL/well into the anti-CD3 prebound plates. For cytokine studies, plate the cells in complete media at 2 x 106 cells/mL/well into sterile flat-bottom, tissue-culture-grade 24-well plates for ConA stimulation. For anti-CD3 stimulation, plate the cells in complete media at 2 x 106 cells/mL/well into the anti-CD3 prebound plates.

26. Treat specified wells with various final concentrations of cannabinoids (0 |M, 0.01 |M, 0.1 |M, 1 |M). Using a repeater pipetor, treat the 24-well plates with 10-|L

aliquots of the cannabinoid reagents (or 10 |L absolute EtOH as the non-cannabi-noid-treated control), and treat the 96-well plates with 1 |L of the cannabinoid reagents (or 1 | L absolute EtOH as the non-cannabinoid-treated control). Set up the wells in the 24-well plates in triplicates. Set up the wells in the 96-well plates in quadruplicates.

27. After cannabinoid treatment, incubate the plates for 1 h (in the case of ConA stimulation) in a humidified 5% CO2 incubator at 37°C. After 1 h of incubation, treat specified wells with ConA to a final concentration of 2.5 |g/mL. Treat each well of the 24-well plates with 10 |L of ConA (or 10 |L RPMI as the non-ConA treated control). And treat every well of the 96-well plates with 1 | L of ConA (or 1 | L RPMI as the non-ConA-treated control).

28. Incubate the plates at 37°C in a humidified 5% CO2 incubator 24, 48, and 72 h.

3.1.3. Macrophage Cell Cultures

1. A 3% thioglycolate broth is prepared by dissolving 1.5 g Bacto-Thioglycollate dehydrated medium (Becton Dickinson, Franklin Lakes, NJ) in a 100-mL sterile glass bottle containing 50 mL of deionized water. The bottle is placed in a boiling water bath to ensure complete dissolution of the powder. Autoclave the solution at 15 psi, 121°C, for 15 min. Allow the thioglycolate broth to cool to 37°C. Upon cooling, inject 3 mL of the broth intraperitoneally using a 5 mL syringe and 27-gauge needle. Injections should be administered in the lower left section of abdominal area to avoid intestinal or internal organ puncture.

2. Four days following injection, sacrifice the animals via CO2 asphyxiation. Peritoneal macrophages are obtained by peritoneal lavage with 9 mL of cold, sterile 0.95% NaCl. For experimental purposes, put the solution on ice in order to minimize macrophage activity prior to culturing. To do the lavage, make a slight cut in the abdominal area and pull back the fur to expose the peritoneum. Inject the cold lavage medium with a 20-gage needle at the fatty spot on the left side of the animal. Massage the inflated abdominal area and incubate for 5 min to obtain a maximum yield of macrophages. Place the animal on a 50-cc centrifuge tube and use a 20-gage needle to pierce the right ventral lateral section of the peritoneum, allowing the fluid to drain directly into the tube. Pool macrophages from CB2R+/+ and CB2R-/- genotypes in order to obtain higher cell counts.

3. Pooled cells are then filtered through a 70-| m nylon mesh filter screen to remove chunks of tissue and cell debris. Gently centrifuge for 5 min at 327g and 4°C. Pour off the supernatant and lyse the red blood cells for 1 min with 1 mL ACK lysing buffer. After 1 min add 19 mL of PBS to stop the lysis of red blood cells and to maintain macrophage integrity. Pellet cells for 5 min at 327g and 4°C. Pour off the supernatant and resuspend in complete medium.

4. Determine cell viability using trypan blue exclusion dye.

5. Prior to plating the thioglycolate-elicited macrophages, round cover slips are sterilized with ethanol and flamed. Subsequently, the cover slips are added to the bottom of Costar 12-well cluster microplates (Corning, Inc., Acton, MA).

Macrophages are then plated, 1 mL per well, at 0.5-1 x 106 cells per well and allowed to adhere to the topside of the cover slips for 1 -2 h.

6. Adherent cells are then rinsed with 1 mL of complete medium for enrichment. Cannabinoids (0, 0.01 |M, 0.1 |M, 1 |M) are then added to plated macrophages for 1 h.

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Responses

  • Robert
    How to culture splenocyte?
    8 years ago
  • Iona
    How to prepare 3% thioglycollate?
    8 years ago

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