Because of the variability in the level of human cell chimerism after the transplantation of CD34+ cells, the injected mice were pair-matched before the proper experiments. Seven weeks after transplantation, 50 μl of the peripheral blood was collected from the tail vein and placed in the EDTA-containing tubes. Next, the total cell count was evaluated (by using Bürker chamber and Türk’s solution) and the percentage of human CD45+ cells was analyzed by flow cytometry after staining with anti-CD45 PE antibody (BD Biosciences). Animals with chimerism varying less that ±2 % were matched into pairs and subjected to experimental endotoxemia or CLP models. Eight weeks after the transplantation, the selected paired mice were subjected to endotoxemia, receiving an intravenous injection of 40 μg of LPS from E. coli O55:B5 (Sigma-Aldrich) in 100 μl of 0.9 % saline or 100 μl of saline as control.
Anti cd45 pe antibody
Manufactured by BD
The Anti-CD45 PE antibody is a laboratory reagent used to identify and quantify cells expressing the CD45 antigen. It is a fluorescently labeled monoclonal antibody that binds specifically to the CD45 surface protein, which is expressed on the majority of human hematopoietic cells.
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2 protocols using anti cd45 pe antibody
1
Evaluation of Human Cell Chimerism in Sepsis Murine Models
2
Isolation and Stimulation of NK Cells for Degranulation Assay
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To analyze the NK cell degranulation, peripheral blood mononuclear cells (PBMCs) were freshly isolated from buffy coat leukocyte concentrates obtained from anonymous healthy human donors using Ficoll, Histopaque-1077 (Sigma) 27 . Then, NK lymphocytes were isolated from PBMCs by bead magnetic separation. Briefly, the PBMCs were incubated with anti-CD56/biotin antibody (Invitrogen), and then magnetic anti-biotin microbeads were added (Miltenyi). Finally, cells were isolated using a MS-column (Miltenyi) in a miniMACS Separator. After three washes with wash solution (DPBS+ 0.1% albumin + 2mM EDTA), the NK cells were eluted and cultured in RPMI 1640 (Gibco) + 10% FBS. The purification efficacy was analyzed by FC using the following surface markers: anti-CD45/PE antibody (BD Bioscience), anti-CD56/BB515 antibody (BD Bioscience) and anti-CD3/PE antibody (Invitrogen), 1:50 dilution. Once NK cells were isolated, they were incubated with 2 μL rIL-2 (100 U/μL) overnight, to stimulate cellular growth. After 24hs, target cells were co-cultured 1:1 with NK cells in a medium containing 2 μM monensin (eBioscience); cell stimulation cocktail (eBioscience), and anti-CD107a/PE antibody (Invitrogen). After of 4 hs incubation, NKs from all conditions were washed and stained with anti-CD56/BB515 antibody and then analyzed by flow cytometry.
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