Millex sterile syringe filters

Lentivirus was prepared by co‐transfecting HEK293T cells with Lentiviral expression plasmids : psPAX2 : pMD2.G in a 5 : 3 : 2 ratio using polyethyleneimine Linear (MW25,000). Here, psPAX2, pMD2.G, and lentiGuide‐Puro were obtained from AddGene (Cambridge, MA, USA). Supernatants that contained lentivirus particles were collected twice between 36 and 72 h, and lentivirus particles were concentrated by centrifugation with PEG‐6000 (Sangon). Before use, lentivirus particles were resuspended in the serum‐free defined medium and filtered through 0.22 μm Millex® sterile syringe filters (Millipore, Burlington, MA, USA).

Leachates were prepared from six different clay samples as follows: ((1) Clay A—Redmond Healing; (2) Clay B—Kaolinite; (3) Clay C—Red Clay; (4) Clay D—Premium Nutri Clay; (5) Clay E—Green clay; and (6) Clay F—Mirador Chimaque; (Table 1). An adaptation of the British Standard (BS EN 12457 Part 2) “characterisation of waste leaching test” [33 ] was employed. In brief, 20 mg (instead of 100 mg) of clay was weighed out to an accuracy of ±0.1 mg and placed into a sterile 15 mL Falcon tube (with a screw cap; Fisher Scientific, Loughborough, UK). The tube was filled with double-distilled water (Cole-Parmer, St Neots, UK) up to 10 mL (instead of 1000 mL), establishing a liquid-to-solid concentration of 2 mg/mL. The capped Falcon tubes were secured into a variable speed rotatory agitator for 24 h at 7 RPM. The leachate was filtered at the time of extraction by drawing 8 mL of leachate through a 0.45 μm cellulose membrane filter (Millex™ Sterile Syringe Filters; Millipore, Watford, UK) using a 10 mL sterile syringe (Cole-Parmer, St Neots, UK). The remaining 2 mL containing the clay particle sediment was retained for use in EM, whereas the filtered 8 mL was utilized for ICP-MS (Inductively Coupled Plasma-Mass Spectroscopy) and in microbiological and wound healing assays. Leachates were autoclaved and stored in the dark at 4 °C to reduce risk of microbial interference.