Amicon ultra 100 kda filter

The disrupted cell solution was applied to Amicon Ultra 100-kDa filters (Merck) and centrifuged at 6000 × g for 30 min at 4 °C. The upper phase and lower phase of the filter were adjusted to a concentration 1 µg/µL protein by PBS and assayed for cell cluster formation activity for Fig. 1b. Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA).

For nanobody-split Cre construction, GBP1 and GBP6 (Addgene #50791 and #50796, Tang et al., 2013 (link)) were fused with NCre and CCre from split Cre (gifts from Hirrlinger et al., 2009 (link)). AAV purification was performed as described previously (Zolotukhin et al., 1999 (link)). Since AAV serotypes can show tropism for specific cell types, we used a cocktail of 4 serotypes (2/1, 2/5, 2/8, 2/9). After iodixanol step gradient, the virus solution was dialyzed and concentrated with Amicon Ultra 100k Da filters (EMD Millipore, Billerica, MA) with lactated Ringer. Virus copy number was quantified with real-time PCR. Virus titers were in the range of 10^12-14 gene counts/ml.

To confirm EV integrity inside the agarose spots, whole or lysate iWJ-MSC-EV were stained and analysed by super-resolution microscopy. Specifically, iWJ-MSC-EV were stained with DPPE-Abberior STAR RED. Briefly, 4 µL of dye were mixed with 1 mL of diluent C. Then, EVs were mixed 1:1 with the diluted dye and incubated for 5 min with periodically mixing. After incubation, EVs were washed 3 times with PBS using an Amicon Ultra 100 kDa filter (Merck Millipore). Finally, lysate EVs were obtained by RIPA (1x) digestion on ice and periodically vortexing.
Super-resolution analysis of EV structure was performed using Abberior Infinity microscope (Gottingen, Germany) equipped with a 60 × /1.42 NA oil immersion STED objective. STED images of EVs stained with DPPE-Abberior STAR RED were acquired 640 nm excitation line and Abberior STAR RED was depleted with a donut-shaped 775-nm pulsed STED laser.
Under these conditions, approx. 40 nm lateral resolution (full-width-at-half-maximum, FWHM) was achieved for Abberior STAR RED acquisition channel (estimated from fluorescent bead calibration measurements). STED images were acquired with following parameters: pinhole size: 1 AU; dwell time: 5 μs/pixel, XY pixel size: 20 nm. Following acquisition, confocal and STED images were thresholded and smoothed (Gaussian blur, radius = 1.0 Sigma) using Fiji (ImageJ distribution) software.

Isolation of EVs was performed using size-exclusion chromatography. Different columns were used for cell culture supernatants and human samples. The column used for the cell culture supernatants was packed with 10-ml Sepharose CL-2B (GE Healthcare Life Sciences) with a molecular weight separation range of 70 × 10³–40 × 10⁶ (29 ). The eluted fractions (6, 7, 8, 9, and 10; 0.5 ml each) were used in the subsequent steps of this experiment. The loaded samples were prepared by sequential centrifugation. The cell culture media were collected after 48 to 72 h of cell growth (10 × 10⁶ cells in 50 ml) and centrifuged at 500g for 10 min at 4 °C and then at 5000g for 30 min at 4 °C, and finally at 10,000g for 30 min at 4 °C. The supernatant was concentrated using an Amicon Ultra 100-kDa filter with a molecular weight cut-off of 100 kDa (Merck Millipore) according to the manufacturer's instructions. For the human samples, EVs were isolated using a chromatography-based method developed by our group (30 ).