Rat anti mouse cd16 cd32 monoclonal antibody clone 2.4g2

Blood samples were collected from anesthetized mice 1 h after histone injection (60 μg·g⁻¹), after which they were mixed with EDTA‐2K (Dojindo Molecular Technologies, Kumamoto, Japan) and used for platelet counts. Custom platelet count measurements were conducted by Sanritsu Zelkova Co. Ltd. (Kanagawa, Japan) using whole blood samples.
Blood samples were mixed with 3.13% (w/v) sodium citrate (Sigma‐Aldrich) and used for flow cytometry. PLAs were determined as previously described [23 (link)]. Briefly, each blood sample was mixed with PE‐conjugated rat anti‐mouse CD45 antibody (clone 30‐F11; BioLegend, San Diego, CA, USA) and BV421‐conjugated rat anti‐mouse CD41 antibody (clone MwReg30; BioLegend), or isotype control. To block non‐specific binding and remove dead cells from the analysis, each mixture was added to a rat anti‐mouse CD16/CD32 monoclonal antibody (clone 2.4G2; BD Biosciences, Franklin Lakes, NJ, USA) and a Zombie NIR™ Fixable Viability kit (BioLegend). The mixture was then incubated for 15 min at room temperature (18–25 °C) in the dark. After fixation with FACS lysing solution (BD Biosciences), the number of CD45⁺CD41⁺ cells was determined using an LSR Fortessa™ X‐20 system (BD Biosciences). Finally, the percentage of PLAs was calculated using flowjo software (Tree Star Inc, San Carlos, CA, USA).

To prevent non‐specific antibody binding, cells were incubated for 10 min at 4°C with rat anti‐mouse CD16/CD32 monoclonal antibody, clone 2.4G2 (BD Pharmingen), prior to staining with epitope‐specific antibodies for 30 min at 4°C. Antibodies used for flow cytometry are listed in Appendix Table S4.
To exclude non‐viable cells, Zombie Aqua, Zombie Red (Biolegend, San Diego, CA, USA), 7‐amino‐actinomycin (7‐AAD; BD Pharmingen), or Sytox Green (Invitrogen) were used according to the manufacturer's instructions. Cells were analyzed using FACS Calibur or LSRFortessa and sorted using FACS Aria IIIu equipped with the CellQuestPro or FACSDiva softwares (BD Pharmingen). Data analysis was performed using FlowJo software (Tree Star Inc., Ashland, OR, USA).