CD34 PerCP-Cy™5.5

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	samples were incubated in the dark with the appropriate combination of monoclonal antibodies during 15 min. Samples were incubated for 15 min in the dark using an 6-color combination, set up with the following monoclonal antibody panel: 10 μl of anti-CD34-PerCPCy5.5	A total of 100,000 events were acquired (at low speed). Data were analyzed using the Infinicyt program (Cytognos, Salamanca, Spain)

Protocol tips
samples were incubated in the dark with the appropriate combination of monoclonal antibodies during 15 min. Samples were incubated for 15 min in the dark using an 6-color combination, set up with the following monoclonal antibody panel: 10 μl of anti-CD34-PerCPCy5.5
Downstream tips
A total of 100,000 events were acquired (at low speed). Data were analyzed using the Infinicyt program (Cytognos, Salamanca, Spain)

Publication protocol

"The following monoclonal antibodies (mouse anti-human) were used for flow cytometric immunophenotyping of hMSC-EV and hMSC: CD90 FITC (Fluorescein isothiocyanate), CD34 FITC, CD44 PE (phycoerythrin), CD73 PE, CD14 PE, CD63 PE, CD166 PerCP-Cy 5.5 (phycoerythrin-cyanine 5.5), CD34 PerCP-Cy5.5, CD45 PerCP-Cy5.5, HLA-DR PerCP-Cy5.5, CD19 PerCP-Cy5.5, CD81 APCH7 (APC-cyanine tandem dye), CD45 V500 (BD Horizon V500), CD44 APC (Allophycocyanin) and CD105 APC. All of them were purchased from BD Biosciences (San José, CA) except for CD44 APC, Cytognos (Salamanca) and CD105 (R&D Systems, Minneapolis, MN, USA) purchased from Cytognos and R&D systems, respectively. We used a FACSCalibur flow cytometer (BD Biosciences) for hMSC sample acquisition. The FACSCalibur flow cytometer is a 4-color instrument with two lasers, blue (488-nm) and red (633 nm). Labeled cells were acquired immediately after the staining using a FACSCalibur flow cytometer equipped with the CellQuestTM program (BD Biosciences).

Human MSC were characterized using the following conjugated monoclonal antibody combinations: CD34 FITC/CD73 PE/CD45 PerCPCy5.5/CD105, CD44 FITC/CD14 PE/CD19 PerCPCy5.5 and CD90 FITC/CD166 PE/HLA-DR PerCPCy5.5. Human MSC (5 × 105 cells) were suspended in PBS and incubated with combinations of the monoclonal antibodies described above and also was acquired hMSC unstained used as control.

We used a FACSCanto II flow cytometer (BD Biosciences San Jose, CA) for EV acquisition using the FACSDiva 6.1 software (BD Biosciences). The FACSCanto II flow cytometer is an 8-color instrument with 3 lasers, blue (488-nm, air cooled, 20-mW solid state), red (633 nm, 17-mW HeNe) and violet (405 nm, 30-mW solid state).

For FACSCanto II flow cytometer calibration, we used SPHERO™ Rainbow Calibration Particles (Rainbow Calibration, eight peaks-Spherotech, Inc. Lake Forest, USA), following the recommendation of the EuroFlow consortium [50], with adaptation of light scatter detectors channels to properly identify the EV. For fluorescence compensation, BD™ CompBeads (BD Biosciences, San José, CA, USA) were used with generic and with the fluorochrome-label antibodies used in our experiments. Samples were acquired after daily evaluation of instrument’s performance using the Rainbow beads particles. These particles were also used to identify the electronic noise or background, which comes primarily from extraneous particles reaching the detector (light scatter) [9]. The samples were acquired after the cytometer was calibrated and compensated.

Before EV acquisition, the instrument was washed with rinse solution and with double filtered PBS (through 0.2 μm membrane Millipore filters) after 6 h of sedimentation, to reduce the instrument background noise (Fig. ​(Fig.1).1). The forward scatter component (FSC) parameter is used to determine the EV size. Double-filtered PBS was acquired with a mix of fluorescent beads composed of varied diameters (0.5, 0.9 and 3 μm) Megamix (Biocytex, Marseille, France) and perfect-Count Microespheres (Cytognos, Salamanca, Spain) of 6–6,4 μm in size. The beads of different sizes were used in all of the experiments, with this approach we validated our instrument basis on the capacity to discriminate between 0.5 and 0.9 μm Megamix beads using the FSC parameter, as well as their background noise. The acquisition of the samples was only performed when the number of double-filtered PBS events acquired per second ranged between 25 and 50 at low speed with threshold settings between 300 and 350. EV recovered from ultracentrifugation were suspended in double filtered PBS and stained by direct immunofluorescence using monoclonal antibodies. For the study of antigen expression, samples were incubated in the dark with the appropriate combination of monoclonal antibodies during 15 min. For the optimization of the immunophenotypic characterization of EV released from hMSC by FCM, in the immunophenotypic panel hMSC markers (CD90, CD44 and CD73), negative markers of hMSC (CD34 and CD45) and EV markers (CD81 and CD63) were included. Samples were incubated for 15 min in the dark using an 6-color combination, set up with the following monoclonal antibody panel: 5 μl of anti-CD90-FITC/ 10 μl of anti-CD73-PE or anti-CD63-PE/10 μl of anti-CD34-PerCPCy5.5/5 μl of anti-CD44-APC/ 5 μl of anti-CD81-APCH7/5 μl of anti-CD45-V500. After incubation the excess of antibodies was washed with double filtered PBS at 2000 g for 10 min. The final volume (suspended cells, MoAbs, PBS and 500 μl Megamix beads) was 700 μl.
To analyze if the antibodies indicated above do not form aggregates we used as negative control the double-filtered PBS containing each single antibody as well as the combination of all the antibodies used. The doubled filtered PBS was stained following the same methodology as EV (Additional file 1: Figure S2 and Additional file 2: Figure S3). Data were acquired immediately after the staining. In all samples, EV not labeled with the different antibodies were used to discriminate the positive and the negative population.

A total of 100,000 events were acquired (at low speed). Data were analyzed using the Infinicyt program (Cytognos, Salamanca, Spain)."

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