Es 20

The MTT assay started 24 h after the medium supplemented with nanoparticles was added to 24 well tissue culture test plates seeded with MSC and has been performed every day during the incubation period (n = 3). The culture medium was replaced by 1 ml of 2.5 mg/ml MTT (Sigma, UK) solution prepared in DMEM/Ham`s F-12 medium (Sigma, UK), followed by two hours incubation at 37 °C with 5% CO₂. After incubation, the MTT solution was replaced by 1 ml of 99,8% isopropanol (STANCHEM, Poland). The plates covered with tinfoil have been shaken for 15 min at 100 rpm (ES-20, Biosan), followed by the color change quantification using the plate reader (Synergy H1, BioTek) at 570 nm.
The cell viability was assessed by the formula: $Cellviability\left(\%\right)=\frac{\left({\text{O}\text{D}}_{570}\text{o}\text{f}\text{t}\text{e}\text{s}\text{t}\text{n}\text{a}\text{n}\text{o}\text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e}\text{s}\right)–\left({\text{O}\text{D}}_{570}\text{o}\text{f}\text{b}\text{l}\text{a}\text{n}\text{k}\right)}{\left({\text{O}\text{D}}_{570}\text{o}\text{f}\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)–\left({\text{O}\text{D}}_{570}\text{o}\text{f}\text{b}\text{l}\text{a}\text{n}\text{k}\right)}x100\%$

Hemoglobin (Sigma‐Aldrich) was dissolved in 0.1 M phosphate buffer (PB) (pH 7.4) to 30 mg/mL. Each chemical (glycidol, epichlorohydrin, 3‐MCPD, glyceraldehyde, acrylic acid, and 1,2‐propanediol) was dissolved in 0.1 M PB (pH 7.4) to 1000 mM. Hb (15 mg/mL), and each chemical (100 mM) were mixed in PB (pH 7.4) in a total volume of 500 μL and incubated at 37°C for 1–20 days in an orbital shaker‐incubator (ES‐20; Biosan, Riga, Latvia) at 190 rpm (Figure 1a).

Time–kill curves were determined as described in [52 (link)] with some modifications. One colony of each bacterium (Psg CFBP 2214 and Cff CFBP 3418) was pre-cultured in 4 mL of King’s B liquid medium for 12 h at 28 °C and incubated on a shaker ES 20 (Biosan, Riga, Latvia) at 200 rpm. The cells were precipitated via centrifugation and titrated to a concentration of 10⁴ CFU/mL with sterile water. Bacterial titer control was carried out spectrophotometrically according to the OD₆₀₀ index measured using a Nanodrop One (Thermo Fisher Scientific, Waltham, MA, USA). The cell suspension was then transferred to 1.5 mL sterile test tubes, and preparations were added to concentrations of 1xMBC. After that, the tubes were placed in a Thermomixer C (Eppendorf, Hamburg, Germany) and cultivated at 27 °C and 350 rpm. After 0, 2, 5 and 30 min and 1, 2 and 24 h, 10 µL of the mixture was taken, diluted in sterile SPS buffer, and dispersed on King’s B agarized medium. Colonies were counted after 48 h of cultivation at 28 °C. A suspension without antimicrobial agents was used as a negative control. The experiment was repeated three times.

One gram of sample
was extracted with 6 mL of an aqueous solution of urea (4 M), ammonium
bicarbonate (0.1 M), and dithiothreitol (0.005 M), as previously reported.^{39 (link)} After 40 min of stirring with a vortex and reciprocating
shaker (Stuart Scientific, Vernon Hills, IL, USA), the samples were
centrifuged (5810R, Eppendorf, Hamburg, Germany) at 3220g for 5 min at 4 °C. One mL of the supernatant was alkylated
by incubation at 50 °C with 25 μL of iodoacetamide (0.5
M) for 20 min. Then, the sample was diluted with 1.64 mL of ammonium
bicarbonate (0.025 M) and added with 200 μL of trypsin (1 mg/mL,
dissolved in HCl 0.001 M, pH 3) and 200 μL of chymotrypsin (1
mg/mL, dissolved in CaCl₂ 0.002 M, HCl 0.001 M, pH 3).
These amounts correspond to an enzyme concentration of 130.5 μg/mL.
Digestion was carried out overnight (18 h) at 37 °C in an incubator
with an orbital shaker (ES-20, Biosan, Riga, Latvia). At the end of
the digestion, the enzymes were inactivated with 500 μL of 10%
(V/V) formic acid (1 mL for soybean meal). Prior to UHPLC/ESI-MS/MS
analysis, samples were centrifuged at 6708g at 4
°C for 10 min and then filtered through syringe filters (0.45
μm). All the samples were prepared and analyzed in duplicate.

Blood of 3 healthy volunteers with vitamin D levels > 30 ng/mL was collected in 13 mL i-PRF+ tubes (PROCESS FOR PRF, 06000 Nice, France) and immediately placed in a centrifuge (“PRF DUO Quattro”). PRF was obtained by centrifugation at 700 rpm for 5 min (for women) or 6 min (for men). After the centrifugation, the upper layer of liquid PRF (1 mL) from one donor of each tube was transferred into a 50 mL tube, and mixed together for further use.
An amount of 0.5 mL of liquid PRF was used to obtain one PRF sample. To prepare PRF samples with CLP (PRF_CLP), 0.5 mL PRF was added to pre-weighed 0.5 mg CLP with an automatic pipette and mixed well with a spatula. Samples for FTIR and SEM analysis were prepared by incubation (Environmental Shaker-Incubator ES-20, Biosan, Riga, Latvia) at 37 °C for 1, 3 and 7 days and then lyophilized for 72 h. For drug release and cell experiments, coagulated PRF and PRF_CLP samples were used.
Written consent from all of the volunteers for use of their samples in the research studies was obtained. All donors were free of any infectious disease and had no abnormal nicotine or alcohol use. None of the subjects used any anticoagulant drugs. Permission No. 6-2/10/53 of the Research Ethics Committee of Riga Stradins University was obtained for the study.

Plasma was removed from the whole blood, and 800 μL of saline was added to the precipitate. Then, the blood sample was centrifuged (1000× g, 5 min) and washed three times. Afterward, it was stored at −80 °C until use. The lysed blood sample (300 µL) was alkalized with 1 M potassium bicarbonate (20 μL), and the internal standard DHP-Val-d7-FTH (10 ppm) was added. FITC (5 mg, 13 μmol) dissolved in N,N-dimethylformamide (30 μL) was added to the mixture, which was then heated at 37 °C for 18 h in orbital shaker-incubator (ES-20; Biosan) with shaking at 190 rpm.

PVA commercial filament and PVA filament prepared by HME (3-m long) were soaked in a saturated solution of IND in methanol at 40 °C for 24 h in a shaker-incubator (model ES-20, Biosan, Riga, Latvia). The filaments were then dried in an oven at 60 °C until they reached a steady weight. Finally, the IND-loaded filaments were kept in a desiccator.