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PE anti-p53 Antibody
645806
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p53 is an important signaling molecule which has versatile roles in different stages of maturation and activation of a cell. We wanted to decipher the expression of p53 in activated T cells in a time dependent manner as we hypothesized that activation of CD8+ T cells will transform it into a more biochemically and energetically higher level, enabling cell multiplication, which will cause down-regulation of p53 expression.
Flow Cytometry
Splenocytes from mouse activated in an antigen-specific manner
Mouse splenocytes were cultured for 3 days in an antigen-specific manner against T cells (TCR specific stimulation). Cells were cultured in 24 well plates, ~106 cells in each well. Duplicate wells were done for each time point and we did a 0 hr.(basal), 24 hr, and 48 hr culture. At the end of each time point, cells from respective wells were washed twice in PBS and stained in a 1:100 dilution of FITC-CD8 and PE conjugated anti-p53 fluorochrome antibody. The first part of staining was done for surface receptor CD8-FITC (1:100 in PBS) and kept for 30 mins on ice. Next cells were washed twice with PBS and permeabilized (intracellular staining for p53) by adding Fixation/Permeabilization solution (BD Biosciences) for 20 minutes at 4 degrees C.
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Next, cells were washed two times in 1X BD Perm/Wash™ buffer. Permeabilized cells were resuspended in 50 uL BD Perm/Wash™ buffer containing a pre-determined optimal concentration (1:100) of a fluorochrome-conjugated anti-p53 antibody. Incubated at 4 degrees C for 30 minutes in the dark, cells were then washed twice with 1X BD Perm/Wash™ buffer (1 mL/wash for staining in tubes and 250 uL/wash final volume for staining in microwell plates) and resuspended in Staining Buffer prior to flow cytometric analysis.
Flow cytometry
Expression of p53 was found to decrease in activated CD8+ T cells with time.
MFI gives an accurate level of expression rather than percentage positive cells
Easy and accurate method of estimating expression of intracellular protein.
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Reliable and repeatable experimental procedure utilizing FACS to measure p53 expression in cells.