Splenocyte and TCell Proliferation Assays

1. The protocol described in the BrdU cell proliferation ELISA kit is used to assess cell proliferation.

2. Forty-eight hours after the cells were stimulated with ConA or plate-bound anti-CD3 antibody, treat each well in the 96-well microtiter plates with 10 |L of bro-modeoxyuridine (BrdU)-labeled stock solution. Return the plates to the same 5% CO2 incubator as described in the kit. After incubating for 24 h more, remove the 96-well microtiter plates containing the BrdU-labeled cells from the incubator. Centrifuge the plates in a plate centrifuge (Heraeus Model no. Megafuge 2.0 R table-top centrifuge) at 300g for 10 min to pellet the cells.

3. Remove the supernatant by suction using a sterile cannula and dry the plates containing the cell pellets in a 60°C oven for 30 min. Proceed as the kit protocol describes to fix the cells, detect the denatured BrdU containing DNA with anti-BrdU-peroxidase antibody, and visualize the BrdU incorporation by the colorimet-ric reaction. The colorimetric reaction is stopped by adding 25 | L of 1 M H2SO4 to each well. Gently tap the plates to allow the H2SO4 to mix thoroughly with the contents of the wells.

4. Within 5 min of adding the stop solution, quantify the colorimetric reaction product by measuring the absorbance of the samples at 450 nm (reference wavelength 690 nm) using the SpectraMax Plus Microplate Spectrophotometer plate reader (Molecular Devices, Sunnyvale, CA). The developing color and the corresponding absorbance values directly correlate to the amount of DNA synthesis, which is an indicator of the number of proliferating cells in the cell culture samples.

5. As seen in Fig. 1, cell proliferation is enhanced in the presence of THC (10-100 nM) in CB2R+/+ cells, but not in CB2R-/- cells. Interestingly, cell proliferation in CB2R-/- cells is greater than in CB2R+/+ cells (Fig. 1). Effects similar to those of THC were observed with WIN55,212-2 and 2-AG on splenocyte proliferation (data not shown).

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