Heparinized peripheral blood was obtained from all patients and healthy controls. All heparinized blood samples were diluted with an equal volume of 0.9% saline, and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Paque (Lymphoprep; Axis-Shield PoC AS, Oslo, Norway) density gradient centrifugation [23 (link)]. Following centrifugation at 800 × g for 20 min at room temperature, PBMCs were collected and washed twice with 0.9% saline. FNA samples were taken from palpable lymph nodes, and lymph nodes cells (LNCs) were suspended in 500 μl of saline. LNCs were centrifuged for 15 min at 1,500 rpm, and the supernatant was discarded. Cells were resuspended in 1 ml of VersaLyse (Beckman Coulter, Marseille Cedex, France) and incubated for 5 min at room temperature in the dark. Following centrifugation, LNCs were washed and resuspended in 0.9% saline.
Ficoll paque lymphoprep
Manufactured by Axis-Shield
Sourced in Norway
Ficoll-Paque (Lymphoprep) is a density gradient medium used for the separation and isolation of mononuclear cells from whole blood or bone marrow samples. It allows the rapid and efficient separation of these cells from erythrocytes and granulocytes.
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3 protocols using ficoll paque lymphoprep
1
Isolation of Peripheral Blood and Lymph Node Cells
2
Isolation and Staining of PBMC
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Heparinized whole blood (25–35 ml) was collected from pSS patients and healthy controls. PBMC were isolated by discontinuous gradient centrifugation using Ficoll-Paque/Lymphoprep (Axis shield PoC AS, Oslo, Norway), and washed 3 times with PBS+0.1% BSA. The isolated PBMC were stained directly for flow cytometry.
3
Measuring NK Cell Cytotoxicity
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NK cell cytotoxicity was measured as described.20 (link) Briefly, peripheral blood mononuclear cells (PBMCs) as effector cells (E) were isolated by density gradient centrifugation using Ficoll-Paque Lymphoprep (Axis-Shield PoC As, Oslo, Norway). The target cells (T) were K562 leukemia cells, which were obtained from China Center for Type Culture Collection, Wuhan, China (GDC037), washed in phosphate-buffered saline (PBS) and stained with DIO. PBMCs and K562 cells were incubated at various E:T ratios in triplicate in 96-well Costar plates (Corning Incorporated, Corning, NY, USA) for 4 h at 37 °C and 5% CO2. After incubation, PI was added to each well to assess K562 cell viability. NK cytotoxicity was analyzed by flow cytometry and calculated as the percentage of K562 cells that were dead.
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