Generation of microglia specific reagents from phage displayed peptide libraries.
The present report concerns the generation of specific markers and the establishment of a selection procedure for microglia specific molecules from phage displayed peptide libraries. Negative selection against a mouse monocytic cell line (IC-21) and positive selection against primary mouse microglia was combined in the selection procedures using a mixture of two random peptide libraries displayed on phage. In a first set of experiments, one clone was selected that bound microglia and IC-21 cells to equal extent, and three clones that bound to unsorted primary microglia to substantially higher levels than to IC-21 cells. In the second series of experiments, microglia and IC-21 cells were mixed and CD45-positive microglia cells were collected using a FACS sorter. From the latter selection series, three clones were found that preferentially bound to microglia cells. The binding of one of the six selected microglia specific phage clones, clone V-1:19, was competed/inhibited in experiments using soluble synthetic peptides corresponding to the binding motif of the phage clone. The specific inhibition to microglia cells by this synthetic peptide was effective in the concentration range of 0.5-20 microM. The preferential binding of clone V-1:19 to microglia like cells was further demonstrated by staining a panel of cell lines and purified primary mouse microglia.
Lundin
Karin
K
Karolinska Institutet, Department of Medicine and Center for Molecular Medicine, Karolinska Hospital, 171 76 Stockholm, Sweden.
Aarum
Johan
J
Appelsved
Lena
L
Johansson-Borg
Anette
A
von Garrelts
Eva
E
Dypbukt
Jeannette
J
Sandberg
Kristian
K
Persson
Mats A A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis*; D001667:Binding, Competitive; D015415:Biomarkers / Q000032:analysis; D036022:Capsid Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D051379:Mice; D017628:Microglia / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D014760:Viral Fusion Proteins
D000954:Antigens, Surface; D015415:Biomarkers; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D019151:Peptide Library; D014760:Viral Fusion Proteins