96 well harvester

Coculture of Mφ with naive spleen cells was performed as follows. Adherent Mφ among peritoneal exudate cells (PECs) from healthy or 8 weeks after STZ-treatment Wt or Mif−/− mice were obtained. Briefly, the Mφ density was adjusted to 5 × 10⁶ cells/mL, and the cells were plated (100 μL) in 96-well flat-bottom plates (Costar, Cambridge, MA, USA). Three hours later, the PECs were washed three times with warm sterile PBS to remove nonadherent cells, and 10 μg of OVA (Worthington, USA) in 100 μL of DMEM supplemented media was added. Three hours later, adherent Mφ were washed three times with warm sterile DMEM to remove excess OVA that had not been phagocytosed. Spleen cells from OVA-transgenic mice were obtained as previously described [45 (link)], suspended at 1 × 10⁶ cells/mL, and added (100 μL) to PECs at a ratio of 1 Mφ : 5 spleen cells. The cocultures were maintained at 37°C in 5% CO₂ for 5 days. Then, [3H]-thymidine (185 GBb/mmol activity, Amersham, UK) was added at 0.5 μCi/well, and the cells were incubated for a further 18 h. The cells were harvested using a 96-well harvester (Tomtec, Toku, Finland) and then counted using a microplate counter (Trilux, Toku, Finland). The values are presented as counts per minute (CPM) from triplicate wells.

Lymphocytes were obtained under sterile conditions from the spleens of naïve or 8 weeks infected BALB/c mice by perfusion, and the erythrocytes in the samples were lysed with Boyle’s solution. The T CD4⁺ cells were specifically extracted using a magnetic separation system MACS coupled to anti-CD4 antibodies (Miltenyi Biotec) according to the manufacturer’s instructions. After determination of viability by Trypan Blue assay, the T CD4⁺ cells were cultured for 2 h in 96-well flat-bottomed culture plates (Costar) previously sensitized with anti-CD3 antibodies (Biolegend). Then, non-stimulated (c), M(SGSTF), M(LPS), or M(Tc-8w) peritoneal macrophages were added in a 1:4 ratio. All co-cultures were incubated for 72 h and then the lymphocytes and supernatants were collected from them. For the proliferation assays, 0.5 μCi of tritiated thymidine (methyl-[³H]-TDR, Amersham) was added and the cells were incubated for a further 18 h. The cells were then harvested on a 96-well harvester (Tomtec), and finally counted using a counter 1450 microβ-plate (Trilux). Values are represented as counts/min (CPM).

CHO-K1 cells stably transfected with Y5R cDNA were plated in 96-well plates, cultured in 1% FBS media for 24 h and treated with a range of NPY concentrations. After 24 h, the number of viable cells was assessed using MTS-based CellTiter 96^®AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI). Additionally, ³H-thymidine uptake under the same conditions was measured. To this end, 0.5 μCi of [³H]thymidine per well was added for the last 6 h of incubation and the cells were harvested in a 96-well harvester (Tomtec, Hamden, CT) and counted in a Wallac 1205 Betaplate Liquid Scintillation Counter (Perkin Elmer, Boston, MA)^{81 (link)–83 (link)}. The effect of NPY (10⁻⁷M) or Y5R antagonist (10⁻⁶M) on cell number was determined by cell count 48 h after culture in various serum concentrations (0, 1, 10% FBS). To assess DNA synthesis in situ, CHO-K1/Y5R transfectants were cultured on glass cover slips, incubated with 10 µM EdU (Thermo Fisher Scientific) for 1 h and stained according to the manufacturer’s protocol. To assess resistance to chemotherapy, ES cells were plated in 96-well or 24-well plates in the standard growth medium, treated with a range of doxorubicin concentrations (0.05–0.4 µg/ml) and cultured for 72 h. The number of viable cells was assessed by MTS assay or cell count upon trypan blue exclusion.

At the peak of EAE, spleens were aseptically removed and single-cell suspensions were prepared by perfusing the spleen with 10 ml of RPMI-1640 media supplemented with 10% FBS, 100 U of penicillin/streptomycin, 2 mM glutamine, 25 mM HEPES, and 1% nonessential amino acids (all from GIBCO, USA). The spleen cells were centrifuged at 1000 g, and erythrocytes were lysed by resuspending the cells in Boyle's solution (0.17 M Tris and 0.16 M ammonium chloride). Following two washes with sterile PBS, the viable cells were counted using Trypan blue exclusion, and the splenocytes were adjusted at 3 × 10⁶ cells/ml in the same medium. The cells (100 μl per well) were seeded in 96-well plates (Costar, USA) and stimulated with 10 μg/ml of MOG35–55. Additionally, the splenocytes were seeded in an anti-CD3-coated plate (10 μg/ml for two hours at 37°C). The proliferation was quantified after five days of incubation at 37°C and 5% CO₂ by pulsing the cells for 24 h with 0.5 μCi [³H]-thymidine (Amersham Biosciences, USA). The cells were harvested on a 96-well harvester (Tomtec, Finland) then counted using a 1450 microβ-plate counter (Trilux, Finland). The values are presented as counts per min (CPM) from triplicate wells. Supernatants from similar cultures without [³H] thymidine were frozen and stored at −70°C until used for detection of cytokines.