Cell Signaling Technology
Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb
9579S
D3E9
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In different healthy and pathological contexts CD4 T cells differentiate into a range of specialized interleukin-secreting subsets. In mouse models of aging and autoimmune diseases we study the plasticity and effector functions of CD4 T helper cells. We used anti-active caspase 3 antibody to test for the presence of apoptotic cells in different cytokine-producing CD4 T cell subsets from old C57Bl/6J mice.
Flow Cytometry
Splenocytes of wild-type 12 months-old C57Bl/6J mouse, incubated in complete culture medium at 37 °C with PMA/ionomycin for 4h and brefeldin A for the last 3 hours.
Surface staining with anti-CD4 Pac Blue in PBS 0.5% BSA incubated 30min at 4°C. Washed twice in PBS and incubated with L/D fixable aqua dye 20 min at 4°C, then washed twice in PBS.
Clone 2.4G2 (Fc block)
Cells are then fixed and permeabilized with commercial kit (30 min at 4°C with fixative, and 15 min at 4°C with permeabilization buffer). Cells were incubated with rabbit anti-active caspase 3 at 1/50 and different anti-cytokine antibodies in permeabilization buffer for 30 min at 4°C and washed once with permeabilization buffer.
Cells were incubated with Al488 anti-rabbit secondary antibody in permeabilization buffer for 30 min at 4°C and washed once with permeabilization buffer and once with PBS BSA 0.5%.
Flow cytometry, blue laser
I could identify active caspase 3 positive cells in the spleen.
The antibody works well.
It is used at high concentration and produces a shift of all cells.
Good option.