21g needle

As a complement to the clinical data observed during the follow-up of patients treated by intra-articular SVF infiltration, we quantified the synovial fluid levels of pro-inflammatory cytokines (IL1β, IL6, and IL8), anti-inflammatory cytokines (IL10), catabolic (MMP2), and anabolic (IGF1) factors, before and 12 months after the treatment. Briefly, 1 to 2 ml of synovial fluid was obtained using a 21G needle (Becton Dickinson). Immediately after extraction, it was frozen at – 80 °C in cryovials (Eppendorf, Hamburg, Germany) until quantification. ELISA tests were carried out following manufacturer’s instructions (all obtained from Wuhan Fine Biotech Co., Wuhan, Hubei, China) and quantified in a Microplate Absorbance Reader (iMark TM, BioRad, Hercules, CA, USA).

Blood samples were collected 1.5 or 24.5 h after the first treatment and were collected by terminally anaesthetising the mice using isofluraneand performing a cardiac puncture with a 21G needle (Becton Dickinson, Franklin Lakes, NJ, USA) and a heparinised syringe. A volume of approximately 600 µl was collected from each mouse into a 1-ml microcentrifuge tube which had been washed with heparin (Eppendorf, Hamburg, Germany). To separate the serum, the samples were centrifuged at 3,000 rpm at 4°C for 15 min and 300 µl of the supernatant was subsequently transferred to a 1-ml microcentrifuge tube (Eppendorf). Sampled were stored at -20°C until analyzed.
Two liver enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were quantified using a UniCelDxC 600 Synchron Clinical System (Beckman Coulter, Brea, CA, USA) following the manufactures recommend procedures and reagents.
Literature values for the normal range in mice were used to minimize the number of animals used. Specifically, the normal range for AST and ALT in mice are reported to be in the range of 300 ± 100 units/L and 100 ± 50 units/L (Oršolić et al., 2010 (link); Gao et al., 2014 (link)).

Kinesin landing rates and surface density calculations were performed using a previously established protocol by Katira et al.^{6 (link)}. Briefly, a flow cell filled with a solution of kinesin diluted in immobilized microtubule BRB80CAT was incubated for 5 min. Next, the flow cell was washed with a 4 µg/mL solution of MTs (sheared three times using a 21 G needle from Becton Dickinson, Franklin Lakes, NJ) in imaging solution with AMP-PNP. The flow cell was sealed with VALAP then immediately imaged. Images were collected every 10 seconds, and the time between MT injection and image collection was measured using a digital stopwatch. The number of landed MTs for each frame were counted using ImageJ RidgeDetection then plotted against the time elapsed after injection of MTs. The kinesin landing rate was determined by fitting the following equation: $$N(t)=N_{Max}\left[1-e^{\left(-\frac{Rt}{N_{Max}}\right)}\right]$$ where N(t) is the number of landed MTs, t is time elapsed after MT injection and R is the kinesin landing rate. Kinesin surface density was calculated using the following equation: $$\rho =-\frac{\ln \left(1-\frac{R}{Z}\right)}{A}$$
where $\rho$ is kinesin surface density, R is kinesin landing rate, Z is the diffusion limited kinesin landing rate assumed to be equal to the landing rate observed at 10-fold dilution from kinesin stock solution, and A is MT area, $A=Lw$ assuming a width w of 25 nm and average length L measured from microtubule images.