Hanks balanced salt solution (hbss)

Healthy adult dairy cows (n = 9) served as blood donors. Animals were bled by puncture of jugular vein and 30 ml blood was collected in 12 ml heparinized sterile plastic tubes (Kabe Labortechnik). Approximately 20 ml of heparinized blood were diluted in 20 ml sterile PBS with 0.02% EDTA (Sigma-Aldrich), layered on top of 12 ml Biocoll® separating solution (density = 1.077 g/l; Biochrom AG) and centrifuged (800 × g, 45 min). After removal of plasma and mononuclear cells, the cell pellet was suspended in 25 ml bi-distilled water and gently mixed during 40 s to lyse erythrocytes. Osmolarity was rapidly restored by adding 4 ml of 10 × Hanks balanced salt solution (Biochrom AG). For complete erythrocyte lysis, this step was repeated twice and PMN were later suspended in sterile RPMI 1640 medium (Sigma-Aldrich). PMN counts were analyzed in a Neubauer haemocytometer. Finally, freshly isolated bovine PMN were allowed to rest at 37°C and 5% CO₂ atmosphere for 30 min until further use.

Primary endometrial epithelial cell culturing was carried out as described before [18 (link),23 (link)]. Briefly, bovine uteri were collected from a local slaughterhouse, and small tissue pieces (approximately 0.1 cm²) from the area between the caruncles were collected. The collected tissue was minced with scalpels and then digested for 2 h at 37 °C in a solution containing 150 U/mL of collagenase, 150 U/mL of hyaluronidase, 200 U/mL of penicillin and 20 μg/mL of streptomycin (Sigma-Aldrich) in Hank’s balanced salt solution (Biochrom). Cells were then centrifuged, and the cell pellet was washed with cell culture medium (DMEM/Ham’s F-12 and 10% FBS, gentamicin and amphotericin B; all from Biochrom). The cells were seeded in 25 cm² flasks at 37 °C + 5% CO₂ for 18 h. During this time, fibroblast cells had already attached, while the medium containing non-attached cells was transferred to a new 25 cm² flask in order to obtain a pure (>99%) epithelial cell culture. Cells were transferred to a 75 cm² flask when a confluence level of >80% was achieved. Cells were allowed to grow and finally passaged into a 24-well plate at a final density of 2 × 10⁵ cells per well.

The LM culture medium was based on RPMI 1640 with phenol red, low endotoxin and without L-Glutamine supplemented with 10% FCS, 20 mM L-Glutamine, and 1000 U/ml Penicillin/1000 µg/ml Streptomycin (both by Thermo Fisher Scientific, Waltham, MA, USA). The LM starvation medium contained the same supplements like LM culture medium but no FCS.
Phosphate-buffered saline supplemented with calcium and magnesium ions (PBS) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). EasyColl and Hanks balanced salt solution containing calcium and magnesium ions (HBSS) were provided by Biochrom (Berlin, Germany). All other chemicals were purchased from Sigma (St. Louis, MO, USA) if not stated differently.

Cell proliferation was determined after 4 days by means of commercial WST-1 assay (Roche, Mannheim, Germany), as previously published [11 (link)]. Briefly, cells were rinsed 3 times with Hanks’ balanced salt solution containing calcium and magnesium (Biochrom) and were subsequently incubated for 30 min with 500 μL WST-1 solution (1:10 dilution with cell culture medium) at standard cell culture condition. Supernatants were collected and 100 μL of each supernatant were transferred to 96-well plates (VWR). Absorbance was measured at 450 nm with a multi-well plate reader (Tecan F200; Tecan, Austria). Cell number was evaluated by standard curve. Experiments were performed at least with 4 replicates.

For investigation of interaction between chondrocytes with T-lymphocytes an adhesion assay was performed as previously described [14 (link)]. Briefly, primary human chondrocytes were grown to subconfluence in monolayer culture and either left untreated alone or co-cultured with T-lymphocytes (Jurkat cells) (1×10⁶/ml), or were treated with 10ng/ml TNF-β, or 10ng/ml TNF-β and 5μl/ml anti-TNF-β, or 10ng/ml TNF-α, or 10ng/ml TNF-α and 5μl/ml anti-TNF-α and then co-cultured with T-lymphocytes for 8h. Additionally, in another set of experiments, primary human chondrocytes were either co-cultured with T-lymphocytes and/or with 5μM resveratrol and/or 10ng/ml TNF-β and/or 10ng/ml TNF-α for 8h, or were transfected with Sirt1-ASO or Sirt1-SO in the presence of Lipofectin (10μl/ml) for 24h prior to co-treatment with T-lymphocytes and 5μM resveratrol and 10ng/ml TNF-β or 10ng/ml TNF-α for 8h. After 8h of co-culture, non-adherent T cells were carefully removed by gentle washing once with Hank´s balanced salt solution (Biochrom, Germany) and photographed under a light microscope (Zeiss, Jena, Germany). The number of adhered T-lymphocyte colonies on chondrocytes was determined by scoring 10 different microscopic fields. This experiment was repeated three times independently and statistical analysis was done to obtain the final values.

Blood sampling for PMN isolation was conducted using healthy adult dairy cows (n = 3) via jugular vein puncture. The heparinised blood was diluted in an equal volume of sterile phosphate-buffered saline (PBS), which contained 0.02% EDTA (Gibco, Dreieich, Germany). It was then layered on 12 mL of Biocoll^® separating solution (Biochrome AG, Berlin, Germany), and centrifuged (45 min, 800× g, room temperature (RT)). The upper layers containing plasma and peripheral blood mononuclear cells were eliminated, and the remaining sediment composed of PMN and erythrocytes was carefully isolated. The cellular sediment was gently re-suspended and shaken in 25 mL of sterile distilled water for 40 s to lyse erythrocytes. Osmolarity was adjusted immediately by adding 2.5 mL of sterile Hanks-balanced salt solution 10× (Biochrom AG). Isolated PMN were then washed twice (400× g, 10 min), resuspended in sterile RPMI 1640 cell culture medium, and counted in a Neubauer haemocytometer chamber according to Fichtner et al. (2020).

Bone marrow was aspirated bilaterally from both the anterior and posterior iliac crests (10 mL/site) from breast cancer patients or healthy volunteers. The following procedures were accomplished under sterile conditions. The bone marrow aspirates were washed in Hanks’ Balanced Salt solution (HBSS) (Biochrom AG, Berlin, Germany), diluted in Dulbecco’s phosphate-buffered saline (DPBS) (Life Technologies), and separated by density centrifugation using Ficoll Paque Plus (GE Healthcare, Munich, Germany). Mononuclear cells were collected from the interphase layer and washed twice in phosphate buffered saline (PBS) with 10% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel). The cytospins were prepared by centrifuging the bone marrow mononuclear cells onto glass slides (Superfrost plus, Glaswarenfabrik Karl Hecht KG, Sondheim, Germany; 7 × 10⁵ mononuclear cells per slide). The slides were air-dried overnight and stored at −80 °C.
For the experiments using peripheral blood from healthy individuals, the PBMC were separated by Ficoll density centrifugation. The obtained PBMC were collected from the interphase layer, washed with PBS, and either applied for the spiking experiments or analyzed by Western blot analysis. For the spiking experiments, the cell lines were spiked into the blood or bone marrow from healthy individuals and processed as described.