This application protocol describes an exemplary procedure, which could be used in order to isolate LOX-1+ cells. Here, LOX-1 cells were isolated from cord blood mononuclear cell suspension from one individual.
The human LOX-1 antigen, the lectin-like oxidized low-density lipoprotein receptor 1, is also known as OLR1 or CLEC8A and belongs to the C-type lectin superfamily. LOX-1 is found on endothelial cells, macrophages, platelets, fibroblasts, and smooth muscle cells. It plays a role in endocytosis, phagocytosis, apoptosis, and inflammatory processes. LOX-1 is expressed on a population of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in peripheral blood of cancer patients1. Moreover, a high density of tumor-associated neutrophils (TANs) expressing LOX-1 and arginase 1 is strongly correlated with a reduced expression of cytotoxic granzyme B and the proliferation marker Ki67 in closely located T cells, suggesting a suppressive function of LOX-1+ TANs2.
When working with anticoagulated cord blood, cord blood mononuclear cells should be isolated by density gradient centrifugation, e.g., using Ficoll-Paque™. For details regarding the preparation of mononuclear cells please refer to the Resources section of the Diamond CD34 Isolation Kit, human.
▲Note: To remove platelets after density gradient separation, resuspend cell pellet in buffer and centrifuge at 200×g for 10−15 minutes at 20 °C. Carefully aspirate supernatant. Repeat washing step.
▲Note: It is strongly recommended to screen biological samples for the presence of LOX-1+ cells before proceeding with the separation protocol.
1.1 Labeling of LOX-1+ cells using LOX-1 Antibody, anti-human, PE, REAfinity™
1.2 Labeling of LOX-1+ cells using LOX-1 Antibody, anti-human, APC, REAfinity
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