Guava 8ht flow cytometer

A panel of 68 miRNAs (listed in Table S1) was used for profiling 40 μL of urine or 15 μL of RNA samples of uEVs isolated by the different methods. For each sample run, Firefly Particles (35 μL) were added to a well of a 96-well filter plate and filtered. Next, 25 μL of Hybe Buffer and 25 μL of sample was added to each well. The plate was incubated at 37 °C for 60 min with shaking. After rinsing, 75 μL of 1× Labeling Buffer was added to each well. The plate was incubated at room temperature for 60 min with shaking. After additional rinses, 65 μL of 95 °C RNAse-free water was added twice to each well to elute the ligated sample. Thirty microliters of this meltoff was mixed with 20 μL PCR master mix. The mixture underwent 27 cycles of PCR amplification followed by six cycles of asymmetric amplification. Next, 60 μL of Hybe Buffer was added back to each well of the original particles, followed by 20 μL of the PCR product, and the plate was incubated at 37 °C for 30 min with shaking. After rinsing, 75 μL of 1× Reporting Buffer was added to each well, and the plate incubated at room temperature for 15 min with shaking. After final rinses, 175 μL of Run Buffer was added to each well. Particles were scanned on an EMD Millipore Guava 8HT flow cytometer. Raw output was background subtracted and subsequently employed in statistical calculations.

A panel of 16 differentially expressed miRNAs with moderate to high expression (miR-10b-5p, miR-194-5p, miR-223-3p, miR-132-3p, miR-144-5p, miR-148a-3p, miR-486-5p, miR-363-3p, miR-199a-5p, miR-16-2-3p, miR-142-3p, miR-34c-5p, miR-129-5p, miR-433-3p, miR-885-5p, miR-346) and six stably expressed miRNAs in sequencing (miR-9-5p, miR-92a-3p, miR-98-5p, miR-101-3p, miR-151a-3p, miR-338-3p) was used for validation. In a 96-well filter plate, Firefly Multimix (Firefly BioWorks, www.fireflybio.com) was incubated with 25ul Hybridization Buffer and 25ul total RNA at a concentration of 1 ng/ul at 37°C for 60 minutes. After rinsing to removing unbound RNA, 75ul of Labeling Buffer was added to each well, and the plate was incubated for 60 minutes at room temperature. Adapted-modified miRNAs were released from the particles using 90°C water, and PCR amplified using a fluorescently-label primer set. PCR product was hybridized to fresh Firefly Multimix for 30 minutes at 37°C and re-suspended in Run Buffer for readout. Particles were scanned on an EMD Millipore Guava 8HT flow cytometer. Raw output was background subtracted, normalized using the geometric mean of the six normalizer miRNAs and log-transformed. LIMMA version 3.20.8 [54 ] was used to calculate significance.

HPAEC grown in 6 well plates were exposed to the un-concentrated conditioned media mixed with EGM2-MV (1:1) for 72 h; media were changed to EBM2 1 h prior to the beginning of the experiment. 4 h after TNFα/vehicle control stimulation, cells were harvested and incubated with fluorescently labeled IgG against CD54/ICAM-1 (eBioscience, San Diego, CA), CD62E/E-selectin (Biolegend, San Diego, CA), and CD106/VCAM (BD) on ice for 30 minutes. Labeled cells were analyzed with Guava 8HT flow cytometer ((EMD Millipore, Billerica, MA).

The breast cancer cell lines, MDA‐MB‐231, MDA‐MB‐361, MDA‐MB‐415, MDA‐MB‐453, MCF7, HCC 1395, HCC 1806, and HCC 1954, were obtained from the ATCC Breast Cancer Cell panel (ATCC 30‐4500K, LGC Standards). Multiple myeloma cell line RPMI 8226 was obtained from LGC Standards and melanoma cell line ESTDAB‐056 was a gift from Prof Federico Garrido, (University of Granada, Spain). All cell lines were cultured in DMEM or RPMI‐1640 supplemented with 5% fetal bovine serum (FBS) and 50 μg/ml kanamycin (all ThermoFisher Scientific). For flow cytometry, cells were harvested from culture flasks with a 1–2 min incubation with trypsin (0.025%)‐EDTA (0.01%) solution at 37° celsius. Cells were then resuspended in culture medium to inactivate trypsin and centrifuged at 300g for 10 min at 4° celsius, then resuspended in PFN buffer (PBS, 2% FBS, 0.1% sodium azide). Cells were stained with primary mouse IgG anti‐HLA‐A, ‐B, and ‐C monoclonal antibody W6/32 [19 (link)] for 20 min at 4° celsius, followed by two washes with centrifugation steps as above in PFN. Cells were then stained with FITC‐anti‐mouse IgG (Sigma‐Aldrich UK, F2012) at a dilution of 1/100 for 20 min, and washed as above. Control cells received second stage FITC anti‐mouse IgG alone. Cells resuspended in PFN and were analysed on a Merck‐Millipore Guava 8HT flow cytometer with a 488‐nm laser using Guavasoft 2.7 software.