Costar 3799

Harvested spleens and lymph nodes were torn apart manually using forceps and subsequently filtered through a 70 µm stranger in order to prepare single cell suspensions. To lyse red blood cells, suspensions containing whole blood and spleens were first incubated with a red blood cell lysis buffer containing 150 mM ammonium chloride (Sigma Aldrich) and 10 mM sodium bicarbonate (Sigma Aldrich) at pH 7.4 for 2 min on ice. After incubation, cells were washed with PBS and plated for staining in microtiter plates (Costar 3799, Corning, Corning, USA). When necessary, cells were first stained with Fc-block (anti-CD16/32, eBiosciences, clone 93, 1:100) for 20 min on ice to prevent nonspecific binding. After washing, cells were stained with different combinations of antibodies depending on the experiment (see Supplementary Table 4 for clones). Viability dyes (Live/Dead fixable Aqua/Violet/Near-Infrared; Life Technologies) were included in antibody panels when necessary. For example, apoptosis was determined by positive staining for fluorochrome-conjugated annexin A5 (Biolegend) with simultaneous exclusion of dead cells determined by Live/Dead staining. Single cell suspensions were analyzed using a BD FACS Canto^TM II (BD Biosciences) and data were analyzed using Flowjo v.10 software (Flowjo, LLC, Ashland, USA).

Ninety-six U shaped well plates (Corning Costar 3799) were used. 24 h before platting the wells were layered with 1% agarose to favor microspheroid formation. 3 × 10⁴ cells/well were used for seeding. Human oral squamous carcinoma cells were incubated for 72 h under an atmosphere of 5% CO₂ at 37°C in a CO₂ incubator.