Pluronic f 108nf prill poloxamer 338

The Pluronic F 108NF Prill Poloxamer 338 (PL 338, molecular weight: 14,600 Da), the Pluronic F 127NF Prill Poloxamer 407 (PL 407, molecular weight: 12,600 Da), and the Pluronic L-81 (L-81, molecular weight: 2,750 Da) were products of BASF Corp. (Ludwigshafen am Rhein, Germany) purchased from Sigma-Aldrich (St. Louis, MO, USA). Levofloxacin (analytical reference material, molecular weight: 361.37 Da), methylthiazolyldiphenyl-tetrazolium bromide (MTT, molecular weight: 414.32), Brain Heart Infusion Agar (BHI Agar), and Mueller Hinton Broth were purchased from Accumedia (Neogen Corporation, Lansing, MI, USA). RPMI-1640 medium, fetal bovine serum, antibiotic/antimycotic solution (with 100 UI·mL⁻¹ penicillin and 100 μg·mL⁻¹streptomycin sulfate), PBS 1x with antibiotic/antimycotic solution, and Trypsin-EDTA were purchased from Cultilab Laboratory Materials for Growing Mobile (Campinas, São Paulo, Brazil). All other reagents were of analytical grade. Deionized water (Purelab Option-Q, ELGA LabWater, High Wycombe, UK) was used for all experiments [36 (link)].

Spatially restricted micropatterned culture substrates were prepared using a modified version of a previously established protocol [20 (link)]. All steps are performed at room temperature inside a sterile biosafety cabinet. 50 μg/mL collagen-I from rat tail dissolved in 2N acetic acid was pulled through a 2x1 cm polydimethylsiloxane (PDMS) stamp with 25 μm-wide lanes separated by 25 μm. The collagen was allowed to adsorb to non-tissue culture treated polystyrene for at least one hour before excess collagen was aspirated, micro-channels rinsed with PBS, and PDMS stamps removed. Substrates were further rinsed with PBS and then blocked with 0.2% Pluronic F108NF Prill Poloxamer 338 (BASF) in water for at least one hour. The substrates were rinsed and stored in PBS at room temperature for no more than one month before use. Control, non-patterned substrates were prepared using a 60 μL drop of collagen-I in place of PDMS stamps.

Maxisorp 96-well plates (Nunc-Immuno plate, Nunc) were coated with 50 mg/ml heparin sodium salt (Sigma) in Tris-buffered saline overnight at 4°C, and washed 3 times with PBS/0.05% Tween 20 (TPBS) before use. Nonspecific binding was blocked with 10 mg/ml pluronic solution (Pluronic F108NF prill poloxamer 338, 30085231, BASF) in PBS for 1 h at 37°C. HRG (100 or 500 ng/ml) in PBS, 0.5% BSA was added to the wells for 1.5 h at room temperature (RT). Radiolabeled HRG was added at a concentration of 10 nM in PBS containing 10 µM ZnCl₂ to the six wells, coated with heparin. Blocking with non-labeled HRG (66 nM) in triplicate wells was initiated 15 min before addition of ¹²⁵I-HRG. Samples were incubated for 1 h at RT, followed by washing 3 times in TPBS. The radioactivity in each well was then measured.