Transwell pet membrane inserts

For the co-culture, 4T1 tumor cells were seeded into Transwell PET membrane inserts with a pore size of 1 µm (Corning, 353104) (6 × 10⁴ cells per insert), and H5V endothelial cells were seeded into a 24-well companion plate (Corning, 353504) (6 × 10⁴ cells per well). After 24 h of incubation, inserts containing 4T1 cells were transferred to a plate with H5V cells to form the transwell chamber. 4PYR or PBS (vehicle) (both 100 μM) were added to the upper chamber, and the cells were incubated for 72 h. Then, the inserts with the 4T1 cells were removed, and the H5V cells in a lower compartment were washed three times with PBS and used for the analysis of extracellular nucleotide metabolism. As a control, a single cell type was seeded into a Transwell chamber and analyzed after incubation.

Calu-3 control and TACSTD2 KO cells were seeded on 24-well Transwell PET membrane inserts with a 0.4 µm pore diameter (Corning Inc., Corning, NY) at a density of 1 × 10⁵ cells/insert in 200 µl of cell suspension in the apical compartment and 500 µl of medium in the basolateral compartment. After 3 days when cells reached confluence, the medium was removed from the apical compartment to create air–liquid interface and cells were cultured for another 11 days. The culture medium was changed every second day.

Primary human brain microvascular endothelial cells (HBMECs) (ScienCell) were grown in complete endothelial cell growth medium (EGM-2MV) (Lonza) while primary human astrocytes (ScienCell) were maintained in astrocyte medium (ScienCell). The in vitro BBB model was established as described here [27 (link)]. Transwell PET membrane inserts (12 mm diameter, 0.4 μm pore size) (Corning) were coated with rat tail collagen type I (15 μg/ml) (Merck Millipore) and fibronectin (30 μg/ml) (Sigma-Aldrich) on the apical side and with poly-l-lysine (20 μg/ml) (ScienCell) on the basolateral side. Inserts were turned upside down, and primary human astrocytes were seeded at a density of 25,000 cells per insert and let adhere overnight. Subsequently, inserts were reversed and HBMECs were seeded, on the apical side, at a density of 200,000 cells per insert. The culture was maintained at 37 °C in 5% CO₂ for 4 days.
Ultra-pure LPS-EK (100 ng/ml final concentration) (InvivoGen) was added into the apical chamber. Upon 24-h exposure, astrocytes were detached from the basolateral side of the Transwell insert using Accutase (Gibco), washed three times with ice-cold PBS (Gibco), and dry-stored at − 80 °C. All the experiments were performed in triplicates.

L6 rat myoblasts and C2C12 mouse myoblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific; #31966021) containing l-alanyl-l-glutamine (GlutaMAX), 10% FBS, and 1% penicillin-streptomycin. Myoblasts were kept at a subconfluent level (< 80%) to prevent the loss of myoblastic component as the cells were passaged. In co-culture experiments where proliferation was studied, myoblasts were seeded in normal growth medium and then switched to low-serum medium (DMEM supplemented with 1% FBS) 24 h before exposing them to SVF cells. SVF cells were added at a 1:1, 1:2, or 1:5 ratio to myoblasts in 0.1 μm PET transwell membrane inserts (Corning; #353104) and co-cultured for up to 5 days. In pharmacological inhibitor experiments, MAP kinase kinase (MEK) inhibitor (25 μM; Calbiochem; #513001), Atropine (10⁻⁵ M, Sigma-Aldrich; #A0132), or Norleual (100 pM, Tocris; #5369) were added to cell cultures 2 h prior to adding the SVF cells. In cases where HGF (PeproTech; #100-39) was used the concentration varied from 5 to 30 ng/ml.

Primary myoblasts were seeded in Bioflex 6-well plates at a density of 3 × 10⁵ cells per well. In parallel, tenocytes with a cell density of 3 × 10⁵ cells were added in PET transwell membrane inserts (Corning, NY, USA) with a pore size of 1.0 or 8.0 μm and kept in a separate plate in the incubator until the experiment was begun. During all experiments, the inserts were placed in indirect co-culture with the myoblasts.