Cd45 pacific orange

Platelet-depleted blood (40 μl) was incubated with E. coli (10⁷/ml) in a rolling incubator at 37°C for 15 minutes. After adding 6.4 μl of the stop solution, the leukocytes were stained with antibodies: CD45-Pacific Orange (catalog no. MHCD4530, Invitrogen™), CD14-PerCP (catalog no. 340585, BD Biosciences), CD15-V450 (catalog no. 48-0158-42, Invitrogen™), CD11b-APC/Fire 750 (catalog no. 101262, BioLegend, San Diego, CA), and CD35-Alexa Fluor 647 (catalog no. 1981978, Invitrogen™) for 15 minutes at 4°C. After lysing the red blood cells with fixative-free lysis buffer (Invitrogen™, catalog no. HYL250), the solution was centrifuged at 250×g, 4°C for 5 minutes. After centrifugation, the supernatant was discarded and the pellet was resuspended in PBSA. The expression of the abovementioned markers was analysed with Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific). Granulocytes were gated as CD15+ population (Supplementary Figure 2), while monocytes were gated as CD15- and CD14+ population (Supplementary Figure 3). The activation of granulocytes and monocytes was evaluated by the expression levels of CD11b and CD35 on their surfaces. Flow data analysis was performed with FlowJo version 10.

Adherent cells at passage 3 were dissociated and resuspended in flow cytometry staining buffer (R&D Systems, Minneapolis, MN, USA) at a final cell concentration of 1 × 10⁶ cells/mL. For surface markers characterization, the following fluorescent monoclonal mouse anti-human antibodies were used: CD73 APC (eBioscience™, Thermo Fisher Scientific, San Diego, CA, USA), CD90 BV510 (BD Biosciences, San Jose, CA, USA), CD105 PE-Cyanine7 (eBioscience™), CD14 PE (eBioscience™), CD34 APC-eFluor 780 (eBioscienceTM), and CD45 Pacific Orange (Thermo Fisher Scientific), as published elsewhere [29 (link)]. Cells were washed twice with 2 mL of flow cytometry staining buffer and resuspended in 500 μL of flow cytometry staining buffer. Fluorescence was evaluated by flow cytometry in Attune NxT flow cytometer (Thermo Fisher Scientific). Data were analyzed using Attune NxT software (Thermo Fisher Scientific).

Adherent DPSCs at passage 3 were dissociated and resuspended in flow cytometry staining buffer (R&D Systems, Minneapolis, MN, USA) at a final cell concentration of 1 × 106 cells/mL. For characterization of surface markers, the following fluorescent monoclonal mouse anti-human antibodies were used: CD29 APC (Thermo Fisher Scientific, San Diego, CA, USA), CD73 APC (eBioscience™, Thermo Fisher Scientific), CD90 BV510 (BD Biosciences, San Jose, CA, USA), CD105 PE-Cyanine7 (eBioscience™), CD14 PE (eBioscience™), and CD45 Pacific Orange (Thermo Fisher Scientific). Cells were washed twice with 2 mL of flow cytometry staining buffer and resuspended in 500 μL of flow cytometry staining buffer. Fluorescence was evaluated by flow cytometry in Attune NxT flow cytometer (Thermo Fisher Scientific). Data were analyzed using Attune NxT software (Thermo Fisher Scientific).