Polypropylene round bottom tube

Biofilm formation assays were performed as previously described (64 (link)). One mL of normalized dilutions of overnight cultures was added to 14 mL polypropylene round-bottom tubes (FALCON) and incubated at 37°C in a static upright position for 24 h. Following incubation, planktonic cells were removed from the tubes by a series of three washes with deionized water. After tubes were air-dried, 1.5 mL of 0.1% Gram crystal violet (Remel) was added and used to stain adherent cells for 15 min. The crystal violet solution was removed and the tubes were washed once more with deionized water three times and left to air dry. Once dry, 1.5 mL of an ethanol:acetone (80:20) solution was added to tubes as a solubilizing agent and left at room temperature for 15 min. OD₅₇₀ absorbance of the resulting solution was measured in duplicate for each tube using a BIO-TEK PowerWaveHT spectrophotometer microreader. Statistical analyses and graphical representations for growth curves, acid resistance, and biofilm formation assays were obtained using GraphPad Prism 9. Data was analyzed using two-tailed unpaired Student’s t test with an alpha value of 0.05.

Mouse epithelial CT26 cells (1 × 10⁶ cells/ml) were cultured with E. coli (1 × 10⁷ CFU/ml) at a multiplicity of 10 bacteria per cell in 14 ml polypropylene round-bottom tubes (FALCON, 352059) at 37 °C with shaking at 60 RPM for 2 h. The cells were smeared onto slides, stained with a three-step staining kit (Thermo Scientific Richard-Allan Scientific), and observed under an Olympus microscope with a DP80 camera.

Undiluted whole blood samples were washed by addition of 3 ml of 0.9% NaCl in polypropylene round-bottom tubes (BD Biosciences) and centrifuged (1000 × g). Then, blood sample was put into a TruCOUNT^™ Tube (BD Biosciences, San Jose, USA) and stained on ice for 30 min with: anti-CD45-APC, anti- HLA-DR-PerCP, anti-CD14-FITC, anti-CD16-PE (BD Biosciences) and anti-PD1-PE-Cy7 (eBioscience) monoclonal antibodies (mAbs). The samples were then treated with FACS Lysing Solution (BD) until erythrocytes were lysed, and the cells were immediately processed in the FACSCanto flow cytometer (Becton Dickinson, San Jose, USA) along with 10.000 of beads per tube. The absolute numbers of CD14⁺⁺CD16^-, CD14⁺⁺CD16⁺ and CD14⁺CD16⁺⁺ monocytes were calculated with reference to the bead count. The percentage and the absolute numbers of PD-1- positive cells were estimated in each of the monocyte subset. Flow cytometric data were analyzed using FlowJo software (Tree Star, Inc, Ashland, OR). The gating strategy and analysis of MO subsets was previously described by us [21 (link)] and others [22 (link)] and is presented on Fig 1.

All measurements were performed on the flow cytometer MACS Quant (Miltenyi Biotec SAS, Paris, France) equipped with three lasers, red laser at 633 nm, blue at 488 nm and violet at 405 nm. All cell samples for flow cytometry analyses were layered into 12 × 75 mm polypropylene round-bottom tubes (BD Biosciences). The samples were run at medium speed, and 10 000 events were collected for each cytometry sample. Compensation beads (BD Biosciences) were used according to the manufacturer’s procedure to compensate for channel cross-talk. All data were acquired and analyzed by means of MACS Quantify analyzer software (Miltenyi Biotec SAS).
In our study, a mixed sample of milk and blood cell suspension (volume 1:1 ratio) was used to validate the analytical scatter pattern. The individual fresh blood and the milk cell suspensions after 30 min at 80°C were used to provide the maximum and the minimum cell viability values, respectively and were prepared as shown below. The milk cell suspensions stored in PBS at 4°C were considered as the reference sample compared to the other physico-chemical treatments applied to the cells.