Stain buffer

To perform the multiparametric analysis of monocytes by fluorescence-activated cell sorting (FACS), the following antibodies were used: CD14 (FITC), CD16 (PerCP-Cy), CD45 (V500), CD274 (PE-Cy7), and HLA-DR (PE), all purchased from Becton, Dickinson and Co. The PBMCs were incubated with stain buffer (BD) with the respective mix of antibodies for 30 min at 4 °C, protected from light. Cells were washed twice with stain buffer, centrifuged at 300g for 5 min and acquired by the FACSCanto II (Becton, Dickinson and Co.). Raw data were pooled on the FACSDiva platform (Becton, Dickinson and Co.), and fine population analyses were performed in FlowJo V10 software (Tree Star, USA). Compensation controls were performed by CompBeads (BD Biosciences, USA), and the acquisition controls were based on the fluorescence minus one (FMO) method. CD45⁺ monocytes were stratified as classical monocytes (CD14⁺CD16⁻), intermediate monocytes (CD14⁺CD16⁺), and non-classical monocytes (CD14^dimCD16⁺). See the analysis plan and FMO controls in Supplementary Fig. 1.

The expression of specific mesenchymal surface markers was analyzed with Human MSC Analysis Kit (Beckton Dickinson), according to the producers’ protocol. Firstly, ASC and DFAT cells in passage 2 were detached from plates by Accutase Cell Detachment Solution (Beckton Dickinson) and suspended in BD Stain Buffer (Beckton Dickinson). The specific antibodies against CD90, CD73, CD105 (positive markers) and CD34, CD11b, CD19, CD45, and HLA-DR (negative markers) were added, and the cells were incubated in the dark at room temperature (RT) for 30 min. After incubation, the cells were washed, centrifuged, and suspended in Stain Buffer (Beckton Dickinson) and then analyzed by flow cytometry FASC Canto II. The obtained results were analyzed by using FlowJo software version 10.7.1 (Becton Dickinson, New Franklin Lakes, NJ, USA).

Cells were detached with Accutase Cell Detachment Solution (Beckton Dickinson) and washed in PBS. Required cell number (1 × 10⁶) was resuspend in cold Stain Buffer (Beckton Dickinson) and used for further flow cytometry analysis. Cell markers were analyzed with Human MSC Analysis Kit (Beckton Dickinson) containing antibodies conjugated with fluorochrome against following antigens: CD73, CD90, CD105 (positive markers), CD11b, CD19, CD34, CD45, and PE (negative markers) (Table 1). Cells were incubated in diluted antibodies in the dark for 30 min. After incubation, cells were washed twice with Stain Buffer (Beckton Dickinson) and resuspend in Stain Buffer. Resuspended cells were analyzed using FACS Canto II (Beckton Dickinson) with FACSDiva Software (Beckton Dickinson) and FlowJo 10 (Beckton Dickinson).
In 3rd and 10th div of 3D culture, spheroids were dissociated and resuspended in Stain Buffer. Before running the sample, cells were filtered through 30 µm filter (Miltenyi Biotec, Bergisch Gladbach, Germany) to avoid dublets. Cells were analyzed to compare the change in size with flow cytometry FACS Canto II (Beckton Dickinson) with FACSDiva Software (Beckton Dickinson) and FlowJo 10 (Beckton Dickinson).

Cells were detached from cell culture dishes, rinsed in PBS, centrifuged, and counted. Cells were washed in BD stain buffer (Becton Dickinson) and resuspended in BD stain buffer with primary antibody (1:250 for ETBR, 1:40 for ETAR, 10 μg/1x10⁵ cells for ECE1) to a concentration of 1x10⁵ cells/100 μl and incubated at room temperature for 45 minutes. Cells were washed twice with BD stain buffer and re-suspended in secondary antibody diluted in BD stain buffer (1:100) and incubated at room temperature for 45 minutes. Cells were washed twice and re-suspended in 200 μl BD stain buffer for flow cytometry analysis. Cells were analyzed on BD Accuri C6 flow cytometer and BD CSampler software (Becton, Dickinson and Company, Franklin Lakes, NJ).