Mix n stain kit

Recombinant CRP was dissolved in DPBS (pH 8.0 with 0.2% BSA). CaCl₂ was added to 50 μL of the CRP solution to a concentration of 10 mM, and the solution was maintained for 1 h to allow for the complete reaction. Unbound calcium ions were isolated using an ultrafiltration kit (Mix-n-Stain kit, Sigma) by centrifugation at 14,000 rpm for 5 min.

CAR T cells were stained with fluorescence-conjugated monoclonal antibodies against CD3 (clone SK7, Biolegend, Heidelberg, Germany), CD8 (clone RPA-T8, Biolegend), CD4 (clone SK3, Biolegend), CD45RO (clone UCHL1, BD Pharmingen, Heidelberg, Germany), CD197 (clone G043H7, Biolegend), CD279 (clone EH12.2H7, Biolegend), TIM-3 (clone F38.2E2 eBioscience, Frankfurt, Germany), LAG-3 (clone 3DS223H, eBioscience) and the CAR-specific anti-idiotype antibody ganglidiomab, provided by H. Lode, Greifswald, Germany, and fluorescence-labeled with the Mix-n-Stain Kit (Sigma-Aldrich, Taufkirchen, Germany) or alternatively with a fluorescein isothiocyanate (FITC)-labeled goat-anti-human Fcγ Ab recognizing the hinge domain (Jackson ImmunoResearch, Pennsylvania, USA, product no. 109-095-098) for the CD19CAR T cells. Samples were fixed with 1% paraformaldehyde (PFA) and acquired directly or not later than 24 h after staining. For each sample, 10 000 cells within the respective gates were analyzed with FACS Diva 9.0, using FACS Celesta flow cytometer (BD Biosciences, Heidelberg, Germany) and FlowJo version 10 (FlowJo, LLC, Ashland, OR, USA).