TY - JOUR
T1 - Optimization and application of a flow cytometric PU.1 assay for murine immune cells
AU - Noel, Greg
AU - DeKoter, Rodney P.
AU - Wang, Quan
AU - Hexley, Philip
AU - Ogle, Cora K.
N1 - Funding Information:
Supported by Shriners of North America Grant Number 86300 .
PY - 2012/8/31
Y1 - 2012/8/31
N2 - PU.1 is a master transcription factor whose levels directly influence hematopoiesis, leukemia, susceptibility to sepsis, and macrophage function. Though measurement of PU.1 levels is important to health and disease, most studies have relied on PCR or western blots to measure the expression of this transcription factor. An accessible, validated assay that could measure PU.1 protein in subpopulations of cells is needed. In this work we present an optimized flow cytometric assay to detect PU.1 in subpopulations of immune cells. Murine myeloid cells were fixed in paraformaldehyde, permeabilized, and then stained with anti PU.1 in the presence and absence of a blocking peptide containing the binding site of the antibody. The bound anti PU.1 was then visualized with a labeled second antibody. Methanol and ethanol were tested for their relative ability to permeabilize cells and detect PU.1. The effect of the procedure upon the ability to detect cellular subpopulations was examined. Relative PU.1 1evels in normal T cells, B cells, monocytes, macrophages, dendritic cells, neutrophils, and progenitors from the spleen and/or bone marrow were determined. Finally, PU.1 levels in proliferating myeloid cells from burn mice were determined. There was a dose dependent increase in the amount of PU.1 detected with increasing amounts of PU.1 antibody that was not seen when blocking peptide was used. Methanol or ethanol gave equivalent results as permeabilization agents, but the latter allowed easier detection of surface antigens when surface staining was performed prior to permeabilization. T cells had little if any PU.1, while B cells had intermediate levels of PU.1, and myeloid cells had high levels of PU.1. Monocytes had higher levels of PU.1 than did neutrophils or spleen macrophages. Plasmacytoid dendritic cells had lower levels of PU.1 than did conventional dendritic cells. Immature myeloid cells had higher levels of PU.1 than did mature myeloid cells. In addition, PU.1 levels were higher in proliferating cells than the corresponding non proliferating cells. Myeloid cells derived from burn mice tended to have higher levels of PU.1 than did unburned, but proliferating cells from burn or sham mice showed no difference in their levels of PU.1. This assay should be a useful addition to the tools used to study the function of PU.1 in health and disease.
AB - PU.1 is a master transcription factor whose levels directly influence hematopoiesis, leukemia, susceptibility to sepsis, and macrophage function. Though measurement of PU.1 levels is important to health and disease, most studies have relied on PCR or western blots to measure the expression of this transcription factor. An accessible, validated assay that could measure PU.1 protein in subpopulations of cells is needed. In this work we present an optimized flow cytometric assay to detect PU.1 in subpopulations of immune cells. Murine myeloid cells were fixed in paraformaldehyde, permeabilized, and then stained with anti PU.1 in the presence and absence of a blocking peptide containing the binding site of the antibody. The bound anti PU.1 was then visualized with a labeled second antibody. Methanol and ethanol were tested for their relative ability to permeabilize cells and detect PU.1. The effect of the procedure upon the ability to detect cellular subpopulations was examined. Relative PU.1 1evels in normal T cells, B cells, monocytes, macrophages, dendritic cells, neutrophils, and progenitors from the spleen and/or bone marrow were determined. Finally, PU.1 levels in proliferating myeloid cells from burn mice were determined. There was a dose dependent increase in the amount of PU.1 detected with increasing amounts of PU.1 antibody that was not seen when blocking peptide was used. Methanol or ethanol gave equivalent results as permeabilization agents, but the latter allowed easier detection of surface antigens when surface staining was performed prior to permeabilization. T cells had little if any PU.1, while B cells had intermediate levels of PU.1, and myeloid cells had high levels of PU.1. Monocytes had higher levels of PU.1 than did neutrophils or spleen macrophages. Plasmacytoid dendritic cells had lower levels of PU.1 than did conventional dendritic cells. Immature myeloid cells had higher levels of PU.1 than did mature myeloid cells. In addition, PU.1 levels were higher in proliferating cells than the corresponding non proliferating cells. Myeloid cells derived from burn mice tended to have higher levels of PU.1 than did unburned, but proliferating cells from burn or sham mice showed no difference in their levels of PU.1. This assay should be a useful addition to the tools used to study the function of PU.1 in health and disease.
KW - ETOH
KW - Flow cytometry
KW - MEOH
KW - Monocytes
KW - Myeloid cells
KW - Neutrophils
KW - PU.1
KW - PU.1GFP mice
KW - Thermal injury
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UR - http://www.scopus.com/inward/citedby.url?scp=84863585237&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2012.05.008
DO - 10.1016/j.jim.2012.05.008
M3 - Article
C2 - 22584156
AN - SCOPUS:84863585237
SN - 0022-1759
VL - 382
SP - 81
EP - 92
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -