Cd14 rmo52

The human 20S immunoproteasome core sample was purchased from Boston Biochem. The human i-20s at a concentration of 1.5 mg per ml was incubated with PKS21004 dissolved in DMF with a molar ratio of 1:250 ratio for 1 h at 37 °C, then the mixture was diluted in 50 mM HEPES, pH 7.6, 100 mM NaCl and 1 mM dithiotreitol to a final concentration of 0.1 mg per mL for cryo-EM study. Antibodies used for flow cytometry are described as below (clone, company): CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD11c (3.9, Biolegend), CD14 (RMO52, Beckman Coulter), CD16 (3G8, Biolegend), CD19 (HIB19, BD), CD38 (LS198–4–3, Beckman Coulter), CD27 (O323, eBioscience), IgD (IA6–2, eBioscience), HLA-DR (L243, Biolegend), Ki67 (Ki-67, Biolegend).

Immediately after arrival of the samples, fresh whole blood from each patient was used to analyze immune populations by flow cytometry (100 μL per antibody panel). EDTA was removed from blood by two washes with PBS 1x. Antibody (clone number in parenthesis) used are: Panel 1 Treg-Tfh: CD4 (OKT4), CD25 (B1.49.9, Beckman Coulter), CD127 (A019D5), CXCR5 (J252D4), PD-1(A17188B), CD8 (SK1); Panel 2 Tmem-Teff: CD4 (OKT4), CD8 (RPA-T8), CD45RA (HI100), CXCR3 (G025H7), CCR7 (G043H7), CCR6 (G034E3); Panel 3 Monocytes: CD14 (RMO52, Beckman Coulter), CD3 (UCHT1), CD11c (3.9), CD16 (3G8), HLA-DR (L243), CD56 (5.1H11), CD19 (HIB19); all antibodies from Biolegend^®, San Diego, CA, US, unless otherwise noted. Antibody mixtures were added to the cells and were incubated for 30 min in room temperature. Subsequently, 1 ml of 1x lysing buffer (BD FACS Lysing solution) was added for 10 min. Cells were then washed with FACS buffer (PBS 1x, 2% FBS, 0.02% sodium azide) and immediately run at BD FACSCanto II flow cytometry. FlowJo^® software (FlowJo LLC, Ashland, OR, US) was used for analysis.

Trypsinized MSCs from P2 to P10 were immunophenotypically characterized by flow cytometry using anti-human monoclonal antibodies (mAb) against CD29 (4B4; Cyto-Stat/Beckman Coulter, Fullerton, CA, USA), CD44 (J173; Immunotech/Beckman Coulter, Marseille, France), CD73 (AD2; BD Biosciences Pharmingen, San Diego, CA, USA), CD90 (F15.42; Immunotech/ Beckman Coulter), CD105 (SN6; Caltag, Burlingame, CA, USA), anti-CD146 (P1H12; BD Biosciences Pharmingen), CD45 (IMMU19.2; Immunotech/ Beckman Coulter), CD14 (RMO52; Immunotech/Beckman Coulter), CD34 (QBend10; Beckman Coulter), CD31 (5.6E; Immunotech/Beckman Coulter), CD19 (J3-119; Immunotech/Beckman Coulter) and HLA-DR (Immu-357, Immunotech/Beckman Coulter), as previously described [6 (link)].
Flow cytometry was also used to study the apoptotic characteristics of MSCs at two representative passages (P2 and P6) by means of 7-aminoactinomycin D staining (7-AAD; Calbiochem-Novabiochem, Nottingham, UK) as previously described [9 (link)]. Results were expressed as proportions of 7-AAD^neg (live), 7-AAD^dim (early apoptotic) and 7-AAD^bright (late apoptotic) cells. Acquisition and analysis were performed in a Cytomics FC 500 flow cytometer (Beckman Coulter, Brea, CA, USA) on a minimum of 10,000 events.