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#0 dbbase_sql->halt(Invalid SQL: select * from kra_comment where pid='154965' and iffb='1' order by id limit 0,10) called at [E:\phpweb\phpwebsite\1011kra\63965966\includes\db.inc.php:54] #1 dbbase_sql->query(select * from {P}_comment where pid='154965' and iffb='1' order by id limit 0,10) called at [E:\phpweb\phpwebsite\1011kra\63965966\comment\module\CommentContent.php:167] #2 CommentContent() called at [E:\phpweb\phpwebsite\1011kra\63965966\includes\common.inc.php:551] #3 printpage() called at [E:\phpweb\phpwebsite\1011kra\63965966\comment\html\index.php:13] 留言点评--晋红兵
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发布于:2019-8-3 17:05:49  访问:122 次 回复:0 篇
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Min at 37 in 5 CO2, coverslips ended up washed 3 times in PBS
Coronin-1-GFP was noticed to speedily accumulate in the nascent phagosome and concentrate in the forefront from the 2‘,3‘-Dideoxyadenosine Epigenetics lamellipodia. Stay pictures ended up acquired each individual 10 s at 37 more than a period of time of 5 min (Determine 8, a and b), utilizing confocal microscopy.Benefits Coronin-1 Accumulates at Phagosomes We in the beginning analyzed the presence and distribution of coronin-1 in Uncooked 264.7 cells, a murine macrophage line. Making use of monoclonal antibodies we verified that these cells categorical coronin-1 (p57) by Western blotting (unpublished info). We then set out to PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27513814 analyze its distribution in the course of phagocytosis. However, this together with other offered antibodies specific for coronin were not capable of detecting the protein at endogenous ranges by immunofluorescence microscopy. As an choice usually means of monitoring coronin-1 distribution, we transiently transfected Uncooked 264.seven cells with full-length coronin-1-GFP. In Dictyostelium, coronin-GFP has long been shown to become completely functional (Maniak et al., 1995), so we predicted which the GFP-tagged chimera would faithfully characterize the distribution in the endogenous coronin-1 in Raw 264.7. Transfected cells have been then incubated with IgGopsonized sheep RBCs, and the place of coronin was examined. Figure 1 shows a series of visuals of the consultant transfected mobile captured immediately after an RBC had sure towards the Fc receptors. Coronin-1-GFP was viewed to rapidly accumulate with the nascent phagosome and concentrate in the leading edge on the lamellipodia. Following only a hundred and eighty s the coronin was observed to speedily dissipate with the phagosome. That is reliable with observations of coronin-1 in differentiated HL-60 cells (Itoh et al., 2002), where coronin was identified to transiently affiliate together with the phagosome.Enhance Receptor-mediated Phagocytosis of RBCsFor C3-receptor-mediated phagocytic index measurements, RBCs had been opsonized with IgM as follows: 100 l of inventory RBCs ended up washed when in PBS and forty l rabbit anti-sheep RBC IgM was included, incubated on a rotator at home temperature for 1 h, and subsequently washed two times in PBS. Fifty microliters of C5-deficient serum was additional and incubated at 37 for 20 min. Raw 264.7 cells had been serum-starved for 2 h in DMEM at 37 in five CO2, activated by 100 nM PMA for 20 min in DMEM, and taken care of with either TAT-WD or TAT- Gal protein. Opsonized RBCs had been washed in PBS three times and overlaid on Uncooked 264.7 cells, and phagocytosis was permitted to come about for 60 min at 37 . External RBCs ended up then lysed making use of hypotonic shock, and Raw 264.seven cells ended up mounted in 4 paraformaldehyde for 1 h. The Phagocytic Index, defined as being the range of phagosomes/macrophage, was calculated by using a period contrast microscope (see Determine 4e) to count the common amount of internalized RBCs for each macrophage.Vol. 16, JulyM. Yan et al.Figure one. Distribution of wild-type human coronin-1-GFP (WTcor-GFP) for the duration of phagocytosis. Uncooked 264.seven cells expressing WT-corGFP were being permitted to bind to IgG-opsonized sheep RBCs at four , and unbound RBCs were washed away. Phagocytosis.
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