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      Gating Hierarchy
      Leukocytes
      Leukocytes Leukocytes
      Protocol & Other Details
      Using most permeabilization buffers and fluorochrome-conjugated antibodies, staining with anti-human CD14 antibody (clone M5E2) following permeabilization does not allow resolution of CD14+ monocytes. Alternative staining protocols may allow resolution of surface marker stains that cannot be resolved when staining after permeabilization. Data shown here were generated using Alternative Protocol 2 (Stain–Fix–Perm). See also data from cells stained for CD14 using the Standard Protocol (Fix–Perm–Stain) or Alternative Protocol 1 (Fix–Stain–Perm).
      Notes
      Human Surface Marker Antibody Concentrations:
      Antibody titration is critical for successful post-permeabilization staining of CD markers and other cell surface antigens. The 1X concentration represents cells stained with the recommended test size for live cell stains (see product Technical Data Sheets for test size information). The 1/4X or 1/16X concentrations represent cells stained with 1/4 or 1/16 the antibody recommended for live cell stains.
      Mouse Surface Marker Antibody Concentrations:
      Antibody titration is critical for successful post-permeabilization staining of CD markers and other cell surface antigens. The 1X concentration represents cells stained with the recommended test size for live cell stains. The 1/4X or 1/16X concentrations represent cells stained with 1/4 or 1/16 the antibody recommended for live cell stains.
      YG PE, YG PE-Cy7, and YG PE-Cy5:
      PE, PE-Cy5, and PE-Cy7 fluorochromes may be excited by either a 488-nm blue laser or a 561-nm yellow-green laser. Since excitation by the yellow-green laser improves PE detection sensitivity, data collected from the 561-nm laser detection array (YG PE, YG PE-Cy5, and YG PE-Cy7) are provided along with data from the 488-nm laser detection array (PE, PE-Cy5, and PE-Cy7) when available.
      Fold-Change Statistic:
      Fold-change of a channel (e.g. pStat5-PE) is calculated as the ratio between the median fluorescence intensity of the stimulated sample (e.g. IL-2) and the median fluorescence intensity of the unstimulated sample (e.g. Control).
      Percentile Distance Statistic:
      Percentile distance is a measure of the spread between the 95th percentile and the 5th percentile of the fluorescence intensity of a channel (e.g. CD3-FITC). The distance is calculated as the ratio between the 95th percentile of a channel and the 5th percentile of a channel in its transformed space (log, linear, arcsinh or biexponential).
      Permeabilization Buffers
      BD Phosflow™ Perm/Wash Buffer I (557885) BD Phosflow™ Perm Buffer II (558052) BD Phosflow™ Perm Buffer III (558050) BD Phosflow™ Perm Buffer IV (560746)

      Perm Buffer IV 1X vs. 0.5X:

       

      Perm Buffer IV (provided as a 10X stock solution) may be used at a 1X or 0.5X concentration. The higher (1X) concentration provides optimal resolution of certain intracellular phosphoprotein stains but might result in increased cell loss and decreased ability to stain certain cell surface markers.

      For Research Use Only. Not for use in diagnostic or therapeutic procedures.

      23-23343-00