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PerCP/Cyanine5.5 anti-mouse TNF-α Antibody
506322
MP6-XT22
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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-TNF-a PerCP Cy5.5 to quantify the number of CD44+ CD4 T memory cells co-expressing TNF-a and IL-21 in 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-CD44 antibody and anti-CD4 Al647 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 are finally incubated with PerCP Cy5.5 anti-TNF-a at 1/225 – 1/300 and PE anti-IL-21 antibody in permeabilization buffer for 30 min at 4°C and washed once with permeabilization buffer and once with PBS BSA 0.5%.
N/A
Flow cytometry, blue laser
I could quantify TNF-a-producing CD44+ CD4 T memory cells in the spleen.
The antibody works well.
Not the brightest option.
Gives satisfactory results, with sufficient separation of the positive population.