Cell proliferation elisa colorimetric kit

Every two weeks, three mice per group were euthanized to isolate spleen lymphocytes under sterile conditions. Then, 1 × 10⁵ cells in 200 μL of RPMI-1640 culture medium (CM) were cultured in each well of 96-well culture plates and stimulated with 5 μg/mL of TsoAg (containing native SAG1 and SAG2) as described previously [3 (link)]. In addition, CM-treated cultures without TsoAg stimulation were conducted and used as controls. The TsoAg-induced lymphocyte proliferation was then monitored using the BrdU (5-bromo-2′-deoxyuridine) Colorimetric Cell Proliferation ELISA Kit (Roche) according to the manufacturer’s instructions. Finally, the stimulation index (SI = OD₄₅₀ values from TsoAg-treated cultures/OD₄₅₀ values from CM-treated control cultures) of each group was calculated as described previously [3 (link), 4 (link)].

Since the head kidney is an important lymphoid organ in fish, in the present study, the lymphocyte proliferation to V. harveyi lysate in the head kidney of immunized grouper was analyzed to evaluate whether protective cell-mediated immunity was induced. Three weeks after the second immunization, three fish per group were sacrificed to collect their head kidney lymphocytes via 34–51% percoll gradient isolation (Sigma, St. Louis, MO, USA) under sterile conditions [21 (link)]. Afterwards, the lymphocytes were cultured in triplicate in 96-well culture plates at a concentration of 2 × 10⁵ cells per well in 200 μl of L-15 culture medium (CM). The cells in each well were stimulated with 20 μg/mL of V. harveyi lysate and incubated for 72 h at 25 °C. Con A (10 μg/mL)- and CM-treated cultures were also, respectively, conducted to use as positive and negative controls. The lymphocyte proliferation induced by V. harveyi lysate was monitored by using the BrdU (5-bromo-2’-deoxyuridine) Colorimetric Cell Proliferation ELISA Kit (Roche) according to the manufacturer’s instructions [21 (link)]. Finally, the stimulation index (stimulation index (SI) = OD₄₅₀ values from V. harveyi lysate-treated cultures or Con A-treated cultures/OD₄₅₀ values from CM-treated control cultures) of each group was calculated as described previously and expressed as the mean ± standard deviation (SD).

Cell proliferation was evaluated by BrdU incorporation using Cell Proliferation ELISA colorimetric kit (Roche Diagnostics) according to the manufacturer’s protocol. A549 cells were transfected with ABCB4-overexpression vector were seeded at a density of 10 × 10³/well in 96-well plates. After 24 h cells were starved in serum-free DMEM for further 24 h. BrdU was added to the cells and incubated for 4 h at 37°C. After removal of culture medium the cells were fixed and anti-BrdU antibody was added followed by the substrate. Finally BrdU incorporation was assessed by an ELISA reader.