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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Flow cytometry (FC) provides a reproducible investigation of cell surface antigens on platelets. The aim of this study was to elaborate appropriate protocols and to compare them with other techniques that have already been published. (1) Venipuncture with tubes containing citrate was better for the preservation of the antigenicity than using ACD tubes. The isolated platelets could not be completely distinguished from detritus and protein aggregates. Therefore a platelet concentration between 10(7) and 10(8)/ml measurement buffer was necessary to obtain a sufficient resolution by FC. (2) Isolation methods using either differential centrifugation or diluted Ficoll-Hypaque as a flotation medium provided platelets of equal purity. The method with Ficoll-Hypaque resulted in a higher number of isolated platelets than differential centrifugation. The demonstration of platelets and their antigens in whole blood without isolation gave good results provided the platelets were not activated. Activation of platelets with 1 NIH-U thrombin/l resulted in the loss of a part of the highly activated platelets because of their aggregation. (3) Comparing different concentrations of paraformaldehyde in PBS, fixation with 1% for 15 min provided the best antigen preservation for most of the antigens investigated. Isolation induced platelet activation. In order to avoid this effect, the whole anticoagulated blood was fixed with 1% paraformaldehyde for 15 min immediately after venipuncture. Then the platelets were isolated using diluted Ficoll-Hypaque. In this way, systemic activation of platelets can be detected with antibodies against glycoproteins which are translocated from the alpha-granules or lysosomes to the cell membrane. These activation markers can be determined on immediately fixed platelets (already in the whole blood) without any interference due to unspecific activation caused by the isolation procedure. (4) Platelet treatment with citric acid at pH 3, in order to remove the antigenicity of HLA-class I molecules, was sensitive to immediate fixation with paraformaldehyde in the whole blood. Fixation after isolating the platelets made it possible to demonstrate antigen stripping, and the free heavy chain, devoid of the beta 2-microglobulin, could be clearly demonstrated. (5) Using standardization beads, the average number of antigenic sites per platelet could be determined for the investigated specificities. It was shown that antibodies which have been directly conjugated or biotinylated and combined with streptavidin-phycoerythrin yielded similar results in terms of the number of antigenic binding sites while unconjugated antibodies in combination with FITC-conjugated anti-mouse-IgG led to overestimation of antigenic binding sites.
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PMID:Standardization of the flow cytometric determination of HLA class I antigens, 'platelet-specific' glycoproteins and activation markers. 776 17

The placenta is a rich source of immunocompetent cells. We have studied the phenotype, number and origin of placental mononuclear blood cells isolated from 32 normal term placentae using 4 color flow cytometry. Respective maternal and cord blood leucocyte preparations were also compared. Placental tissue without extraembryonic membranes was cut into small pieces and divided. One portion was washed extensively with ice-cold PBS. Both tissue portions were disrupted in a blender and cells were dissociated by using a 180 mu sieve. Leucocytes were isolated by Ficoll-Hypaque density gradient centrifugation. Maternal and cord bloods were HLA typed and in cases of HLA-A2 or B7/40 disparity, monoclonal anti-HLA antibodies to these antigens showed that unwashed placental tissue contained 35% maternal and 65% fetal cells. This ratio, however, was not reflected for a given cell phenotype. In comparison, washed placental tissue contained cells of fetal origin only. Both unwashed and washed placental tissue contained fewer CD3 and CD4, but more CD8 cells than maternal and cord blood. Markers of NK cells such as, CD16, CD56, and CD57 showed this cellular phenotype to be 15 times more abundant in the placental preparations than in cord and maternal blood. The quantitative differences between peripheral blood and placental CD8 and NK cells were further explored with an antiprogesterone receptor antibody in combination with anti-CD8, anti-CD57 and anti-HLA-DR. The number of progesterone receptor (PGR) positive cells was three times higher in placental tissues than in cord or maternal blood. These data indicate that the phenotypic frequencies of certain placental leucocytes are significantly different from maternal and fetal peripheral blood. Progesterone and the presence of PGR may be important in the differential retention of placental leucocytes.
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PMID:Phenotypic characterization of normal human placental mononuclear cells. 827 Dec 37