40 μm cell strainer

Comet assays were performed as described [45 ] with some modifications. Briefly, young roots were chopped with a razor blade in 400 mM Tris-HCl (pH 7.5). The nuclear suspensions were filtered through 40 μm cell strainers (Corning) to remove cell debris, mixed with an equal volume of 1% low melting point agarose prepared with PBS, and then spread on microscope slides cooled to 4°C for 5 min to solidify the mixture. Embedded nuclear DNA was unwound at 4°C for 5 min in electrophoresis buffer (1 mM Na₂EDTA and 300 mM NaOH (pH> 13)), and then electrophoresed at 300 mA for 20 min at 4°C. Slides were rinsed 3 times with 400 mM Tris-HCl (pH 7.5) before staining the DNA with 100 μg/ml propidium iodide. Fluorescence was visualized with a Leica SP8 laser scanning confocal microscope equipped with a 40X oil objective, using 488-nm for excitation and emission between 500 and 530 nm.

Peripheral blood mononuclear cells (PBMCs) were isolated from total blood using Histopaque-1077 separation according to the manufacturer’s protocol (Sigma-Aldrich). The PBMCs were collected, washed in PBS, and counted. PBMCs were resuspended in resuspension media (RPMI-1640 (Gibco) supplemented with 2 mM l-glutamine, 100 U mL^-1 penicillin, and 100 μg mL^-1 streptomycin, with 40% fetal calf serum (FCS)) then an equal volume of freezing media (resuspension media supplemented with 30% dimethyl sulfoxide (DMSO, Fisher Reagents)) was added to give final density of 10x10⁶ cells mL⁻¹ in 15% DMSO. The sample was then frozen to -80°C in a Mr Frosty Freezing Container (ThermoFisher Scientific) then stored in liquid nitrogen until further use.
Synovial tissue was collected from RA patients undergoing arthroplasty. The fresh tissue was dissected followed by digestion for 2 hours at 37°C in 1 mg mL⁻¹ Collagenase 1 (Sigma-Aldrich). Debris was removed and cells isolated by sequentially passing through 100, 70 and 40μm cell strainers (Corning). The isolated cells were then frozen and stored in the same way as the PBMCs.

In the MFP platform, after centrifugation of cell suspension for 3 min at 500 g, the supernatant was discharged. As a proof of concept, a final concentration ranging from 5 × 10⁶- 1 × 10⁷ cells ml⁻¹ was prepared and added to the delivery buffer and passed through the 40 μm cell strainers (Corning Inc, NY, USA) to remove cell clumps from the suspension. Accordingly, DPBS, DMEM, and optiMEM were used as a delivery buffer to optimize the experimental conditions. Before loading the cell suspension into the syringes, the delivery biomolecules were added to the solution. Next, the cell suspension was forced through the microengineered porous membranes using a syringe pump (Chemyx Inc, TX, USA) at a flow rate range between 0.5 to 10 ml min⁻¹. First, we used Fluorescein isothiocyanate (FITC) dextran molecules (70 and 2000 kDa) (Sigma Aldrich, MO, USA) as delivery cargoes. Next, to prove the ability of this platform in loading large-sized cargoes, histone H2B-GFP (green fluorescent protein) plasmid DNA (Addgene plasmid # 11,680; RRID:Addgene_11680, MA, USA), kindly gifted by Geoff Wahl, was used as a delivery biomolecule to be loaded into the cells of interest^{28 (link)}. Triplicate sampling was conducted for each data point.