Isolation of LOX-1⁺ cells from cord blood mononuclear cell suspension

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.

• Work fast, keep cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling.
• Volumes for magnetic labeling given below are for up to 10⁷ total cells. When working with fewer than 10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁷ total cells, use twice the volume of all indicated reagent volumes and total volumes).
• For optimal performance it is recommended to obtain a single cell suspension before magnetic labeling. Pass cells through 30 μm nylon mesh (e.g. Pre-Separation Filters (30 μm), # 130-041-407) to remove cell clumps which may clog the column. Moisten filter with buffer before use.
• The recommended incubation temperature is 2–8 °C. Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice may require increased incubation times.

1.1 Labeling of LOX-1⁺ cells using LOX-1 Antibody, anti-human, PE, REAfinity™

1. Determine cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend cell pellet in 96 μL of buffer per 10⁷ total cells.
4. Add 4 μL of LOX-1 Antibody, anti-human, PE, REAfinity per 10⁷ total cells.
5. Mix well and incubate for 15 minutes in the refrigerator (2−8 °C).
6. Wash cells by adding 1–2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
7. Resuspend cell pellet by adding 80 μL of buffer per 10⁷ total cells.
8. Add 20 μL of Anti-PE MicroBeads per 10⁷ total cells.
9. Mix well and incubate for 10 minutes in the refrigerator (2−8 °C).
10. Wash cells by adding 1–2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
11. Resuspend up to 10⁸ cells in 500 μL of buffer.
12. Proceed to magnetic separation.

1.2 Labeling of LOX-1⁺ cells using LOX-1 Antibody, anti-human, APC, REAfinity

1. Determine cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend cell pellet in 96 μL of buffer per 10⁷ total cells.
4. Add 4 μL of LOX-1 Antibody, anti-human, APC, REAfinity per 10⁷ total cells.
5. Mix well and incubate for 15 minutes in the refrigerator (2−8 °C).
6. Wash cells by adding 1–2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
7. Resuspend cell pellet by adding 80 μL of buffer per 10⁷ total cells.
8. Add 20 μL of Anti-APC MicroBeads per 10⁷ total cells.
9. Mix well and incubate for 10 minutes in the refrigerator (2−8 °C).
10. Wash cells by adding 1–2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
11. Resuspend up to 10⁸ cells in 500 μL of buffer.
12. Proceed to magnetic separation.

• Choose an appropriate MACS® Column and MACS Separator according to the number of total cells and the number of LOX-1⁺ cells. 
• Always wait until the column reservoir is empty before proceeding to the next step. 

1. Place column in the magnetic field of a suitable MACS Separator. For details refer to the respective MACS Column data sheet. 
2. Prepare column by rinsing with the appropriate amount of buffer: 
   MS: 500 μL 
   LS: 3 mL 
3. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells. 
4. Wash column with the appropriate amount of buffer. Collect unlabeled cells that pass through and combine with the flow-through from step 3. 
   MS: 3×500 μL 
   LS: 3×3 mL 
   ▲Note: Perform washing steps by adding buffer aliquots as soon as the column reservoir is empty. 
5. Remove column from the separator and place it on a suitable collection tube. 
6. Pipette the appropriate amount of buffer onto the column. Immediately flush out the magnetically labeled LOX-1⁺ cells.
7. (Optional) To increase the purity of LOX-1⁺ cells, the eluted fraction can be enriched over a second MS or LS Column. Repeat the magnetic separation procedure as described in steps 1 to 6 by using a new column.
    

1. Condamine T, et al. (2016) Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients. Sci. Immunol.;1(2):aaf8943. .
2. Si Y, et al. (2019) Multidimensional imaging provides evidence for down-regulation of T cell effector function by MDSC in human cancer tissue. Sci. Immunol. 18;4(40):eaaw9159.