Falcon tube

Three tissue samples of the liver, spleen, and thymus from each chicken group were used for immunophenotyping. A small piece of each tissue was softly macerated and filtered using a 70 μm cell strainer (FALCON-Corning, NC, USA) in a centrifuge tube and centrifuged at 376 × g for 5 min. The supernatant was discarded, and the cells were suspended in 1 mL PBS and mixed by gentle rocking, counted, and cells equivalent to 1 × 10⁶/mL from each sample were transferred to falcon tubes (FALCON-Corning) for staining. Mouse Anti-Chicken CD3-FITC [26 ], Mouse Anti-Chicken CD4-APC [27 (link)], and Mouse Anti-Chicken CD8α-PE [26 ] antibodies (SouthernBiotech, Birmingham, AL, USA) reconstituted according to the manufacturer’s recommendations were used for staining the prepared cells. Forty microliters of each antibody adequate for staining 1 × 10⁶ cells were transferred into each test falcon tube containing prepared cells and incubated at 4°C for 30 min. Then, the cells were washed with PBS (pH 7.4, 0.01 M, 4°C) thrice by centrifuging at 376 × g for 5 min at 4°C each. After washing, the cells were suspended in 500 μL PBS, sorted, assessed, and CD3+, CD4+, and CD8+ phenotypes were determined using flow cytometry on 10,000 live cells using a BD FACSCalibur flow cytometer (BD Biotec., San Diego, CA, USA) [28 (link)]. The data generated were analyzed using the CellQuest software (BD Biotec.).

The long-term evolution experiment under strong genetic drift of lines A and B, each of which represent lines of E. coli K12 strain MG1655, is described elsewhere (Alvarez-Ponce et al., 2016 (link)). Additionally, from the same ancestral colony, another five independent experimental evolution lines were established in liquid Luria Broth media (LB, Conda laboratory, Madrid, Spain). Each population lineage was serially passaged each 24 h by diluting 1:100 into 10 ml of fresh LB medium in 50 ml Falcon tubes (Corning, Mexico DF, Mexico). Population lineages were passaged 85 times (an estimated 561 generations, 6.6 generations per passage) (Figure 2).

Electrospun samples were cut in a rectangular shape (2.5 × 1.5 cm) and placed in sterile falcon tubes inert to gamma radiation (Corning Incorporated, Life Sciences, 836 North St. Tewksbury, MA 01,876).
Gamma irradiation treatment was carried out at applied nuclear energy laboratory (LENA), University of Pavia, using a Cobalt-60 energy source for 7 days to achieve a 25 kGy radiation dose (3.5 kGy per day dose rate). Sterilization was performed at controlled room temperature (25 ± 3 °C). The 25 kGy applied sterilization dose is the one suggested by the EMA guideline on sterilization of medicinal products, active substances, excipients, and primary containers. The sterilization process was validated in a previous work [21 , 22 ].
After gamma irradiation, polymeric electrospun scaffolds underwent shape memory treatment and their shape memory properties were verified and compared to not gamma-irradiated electrospun scaffolds.
Rf% and Rr% values were evaluated using Eqs. (1) and (2). Analyses were performed in triplicate and data reported as average values ± SD.