To circumvent this inadequacy, a blocking strategy is used whereby purified unconjugated antibody is added to the sample first, and then stained with the conjugated form. In repeated analysis, this method routinely allows accurate setting of negative control gates that are more efficient than either using FMOs or isotype controls. However, an isotype control is needed to set the background for CD107a staining, as CD107a antibody or its control must be present during the culture period.
Data from flow cytometric analyses are often reported as percentage of a population bearing that phenotype or function. However, percentage only describes the relative distribution of cells in the sample. An increase or decrease in percentage in the population of interest may be influenced by changes in what is considered the irrelevant cells in the population under analysis. As part of the monitoring process, we acquire data using BD TruCOUNT™ counting beads to determine the absolute frequency of cells per milliliter of culture, as opposed to describing their relative distribution by percentage. In addition, data can be influenced by sample size and event count. BD TruCOUNT™ allows normalization between samples, thereby providing for more accurate sample-to-sample comparisons.
|SURFACE MARKER||DYE / FLUOROCHROME||TARGET|
|CD3||APC-eFluor780||Epsilon chain of TCR complex|
T cell, NK subset
|CD45RA||BV605||Naive T cells|
|CD279||PE-Cy7||Programed Cell Death 1|
|INTRA CELLULAR MARKER||DYE / FLUOROCHROME||TARGET|
|Granzyme b||PE Texas Red||Cytokine|