Ammonium chloride solution

Twenty milliliters of peripheral blood were collected from each participant. The blood was used for mononuclear cell isolation as soon as possible using a Lymphoprep™ Tube (Serumwerk, Bernburg, Germany) following the instructions provided. The red blood cells were removed by treating them with an ammonium chloride solution (eBioscience, San Diego, CA, USA) for 10 min at 25 °C. After hemolysis, the cells were washed twice with cold D-PBS (Fujifilm, Tokyo, Japan), counted, and used as PBMCs for the experiments.

Peripheral blood was collected from each participant in Vacutainer test tubes containing heparin sodium (Terumo, Japan). Peripheral blood was diluted with an equal volume of 0.9% NaCl. Eighteen milliliters of diluted blood was carefully layered over 10 mL of Lymphoprep in a Lymphoprep™ Tube (Serumwerk, Germany), and the tube was centrifuged at 800× g for 20 min at 25°C. After centrifugation, the mononuclear cells were collected from a distinct band at the sample/medium interface as described in the separation procedure. The harvested mononuclear cell fraction was diluted four‐fold with 0.9% NaCl and centrifuged at 400× g for 5 min at 25°C. The supernatant was removed, and the red blood cells were hemolyzed using ammonium chloride solution (eBioscience, USA) for 10 min at 25°C. After hemolysis, the cells were washed twice with cold D‐PBS (Fujifilm, Japan), counted, and used as PBMCs for the following experiments.