Incu shaker

Concentration of zinc was determined by means of ICP-AES method (ICP atomic emission spectrometer Varian 710-ES, Varian, Palo Alto, CA, USA). Calibration curve method (Zn concentration range: 0.1–10 mg/L) was used. Biosorbents were analyzed utilizing scanning electron microscopy (Phenom Pro Desktop SEM, Eindhoven, The Netherlands) with an EDS detector preceded by lyophilization of swollen beads (Christ Alpha 1-2 LDplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). The thermostated shaker (Incu-Shaker, Benchmark, Sayreville, NJ, USA) was used for polysaccharide solutions preparation. Peristaltic pumps (Ismatec Reglo Digital, Cole-Parmer GmbH, Wertheim, Germany) were used for pumping the solution through the column in small scale studies, as well as for polysaccharide solution instilling into cold CaCl₂ during sorbent preparation. The authors designed a large-scale column (internal diameter 240 mm, height 130 cm, volume ~60 L, PVC, transparent) equipped with peristaltic pumps, proper valves, and connectors (enabling drop-fed and reverse-fed column work); the control system as well as automatic sampling station was supplied by PPU Prokal Sp. z o.o. (Gliwice, Poland).

Patient-derived scaffolds were prepared from frozen breast cancer samples as earlier described [11 (link)]. Briefly, tumors were washed in 3 mM sodium azide (G-biosciences), 5 mM Ethylenediaminetetraacetic acid (EDTA; Invitrogen), 3.5 mM sodium dodecyl sulfate (SDS; Applichem) and 0.4 mM phenylmethylsulfonyl fluoride (PMSF; Sigma Merck) for 6 h and later in 3 mM sodium azide, 5 mM EDTA and 0.4 mM PMSF for 6 h. Thereafter, tumors were rinsed with distilled water for 72 h and Phosphate Buffered Saline (PBS; Medicago) for following 24 h. Subsequently, PDSs were sterilized with 0.1% Peracetic acid (Merck) in distilled water for 1 h followed by 1% Antibiotic–Antimycotic (Gibco) in PBS for 24 h. Patient-derived scaffolds were stored in 3 mM sodium azide, 5 mM EDTA until usage. All wash and rinse steps were performed at 37 °C, in a 10L Incu-Shaker (Benchmark) by shaking at 175 rpm.

A 500-ml shake flask containing 100 mL of nutritive broth (BIOXON, Mexico) was inoculated with a 5% (v/v) overnight culture of P. aeruginosa with a starting OD_565nm of 0.012 (nutritive broth). The culture was incubated at 37 °C with shaking at 200 rpm in a Benchmark Incu-Shaker until the optical density at 565 nm reached 0.745 ± 0.020 (corresponding to 1.53 × 10⁸ CFU/mL), indicating that the culture was in mid-exponential phase according to the logistic growth model (Additional file 1: Figure S1). The culture was used as an inoculum for rhamnolipid production.

Primary microglial cells were harvested following previously published methods [64 (link)]. Primary cortical tissue was harvested from pregnant Wistar day 18 rats (Charles River Laboratories). The cerebral cortex were dissected from the embryos and broken up in calcium and magnesium free HBSS with 0.125% trypsin for 10 minutes and then mechanically disassociated with a pipette (~20 strokes). The cells were filtered through a 40 μm cell strainer and plated at 5×10⁶ cells per T75 flask. The cells were cultured in 10 ml of growth media, DMEM media (Life Technologies) with 10% Fetal Bovine Serum (Life Technologies), with the media changed ever 2-3 days for ten days. On the tenth day the media was removed and 10ml of fresh growth media was added, the cap of the flask was wrapped in Parafilm to prevent atmosphere exchange, and the flask was shaken at 210 rpm for 1 hour on a Benchmark Incu-Shaker. After the shaking the media was collected and centrifuged to harvest the suspended microglia. Fresh growth media was added to the flask and the cells were allowed to culture for an additional week for a second shaking, to harvest more microglia. The populations obtained using this method were 90-95% pure for macrophage/microglia cell specific marker (Iba1). The harvested cells were then used for the studies, with no additional passages.