Cytokine Assays

3.3.1. Splenocyte Cytokine Assay

3.3.1.1. BioRad Assays

1. Seventy-two hours postculture, remove the 24-well plates from the incubator and collect 120 |L of supernatant from each well and place into a sterile, flat-bottom, non-tissue-culture-treated 96-well microtiter plate. Store the plate containing the supernatant in the -70°C Revco freezer until it is assayed for specific cytokines using the Bio-Plex cytokine assay as indicated in the kit. The protocol included in

Fig. 1. A9-THC enhances CB2R+/+ splenocyte proliferation, but not CB2R-/- spleno-cyte proliferation. Splenocyte proliferation was determined by BrdU incorporation assay as described in the text. Cells were plated at 2 x 105 cells/0.1 mL/well in complete media. Cells were treated with 2.5 |g/mL ConA and the indicated A9-THC concentration for 72 h. Data are expressed as the mean of quadruplicate samples + standard deviation. Vehicle is absolute ethanol.

Fig. 1. A9-THC enhances CB2R+/+ splenocyte proliferation, but not CB2R-/- spleno-cyte proliferation. Splenocyte proliferation was determined by BrdU incorporation assay as described in the text. Cells were plated at 2 x 105 cells/0.1 mL/well in complete media. Cells were treated with 2.5 |g/mL ConA and the indicated A9-THC concentration for 72 h. Data are expressed as the mean of quadruplicate samples + standard deviation. Vehicle is absolute ethanol.

the kit is followed to assess the concentration of IL-ip, IL-2, IL-4, IL-5, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-y and tumor necrosis factor (TNF)-a in the supernatant.

2. The Luminex 100 microplate reader (Luminex Corporation, Austin, TX) is used to determine cytokine concentration using the Bio-Plex cytokine kit.

Fig. 2. A9-THC enhances CB2R+/+ splenocyte IL-2 cytokine secretion, but not CB2 R-/- splenocyte IL-2 secretion. Splenocyte proliferation was determined by BrdU incorporation assay as described in the text. Cells were plated at 2 x105 cells/0.1 mL/well in complete media. Cells were treated with 2.5 |g/mL ConA and the indicated A9-THC concentration for 72 h. Data are experiments and are expressed as the mean of quadruplicate samples + standard deviation. Vehicle is absolute ethanol.

Fig. 2. A9-THC enhances CB2R+/+ splenocyte IL-2 cytokine secretion, but not CB2 R-/- splenocyte IL-2 secretion. Splenocyte proliferation was determined by BrdU incorporation assay as described in the text. Cells were plated at 2 x105 cells/0.1 mL/well in complete media. Cells were treated with 2.5 |g/mL ConA and the indicated A9-THC concentration for 72 h. Data are experiments and are expressed as the mean of quadruplicate samples + standard deviation. Vehicle is absolute ethanol.

3. As seen in Fig. 2, the CB2R mediates THC-induced splenocyte IL-2 cytokine secretion, because this effect is not seen in the CB2R-/- cells. Perhaps more interesting is the finding that in CB2R-/- splenocytes IL-2 secretions are enhanced over those of CB2R+/+ cells (Fig. 2). Effects similar to those of THC were observed with WIN55,212-2 and 2-AG on splenocyte cytokine secretion (data not shown).

3.3.2. T-Cell Cytokine Assays

1. Twenty-four, 48, and 72 h after the cells are stimulated with ConA or plate-bound anti-CD3 antibody, remove 225 | L of supernatant from each well and place into a sterile, flat-bottom, non-tissue-culture-treated 96-well microtiter plate. Store the plate containing the supernatant in the -70°C Revco freezer until it is assayed for specific cytokines using IL-2 and IL-4 BD Bioscience kits. The protocol included in the kit is followed to assess the concentration of IL-2 or IL-4 in the supernatant.

2. Quantify the colorimetric reaction product by measuring the absorbance of the samples at 450 nm (reference wavelength 570 nm) using the SpectraMax Plus Microplate Spectrophotometer plate reader (Molecular Devices, Sunnyvale, CA).

3. As seen in Fig. 3, the CB2R mediates WIN55,212-2-induced IL-2 cytokine secretion, because this effect is not seen in the CB2R-/- cells. Furthermore, in CB2R-/- T-cell IL-2 secretions are enhanced over those of CB2R+/+ cells (Fig. 3).

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