Enzyme free cell dissociation solution hank s based 1

For preparation of cell lysates, the supernatants were collected following the co-culture experiment, and the cells were detached using Enzyme-Free Cell Dissociation Solution Hank’s Based (1 ×) (Merck KGaA, Darmstadt, Germany) and added to the supernatant. Cells were centrifuged at 800 rcf for 10 min and washed twice with 5mL DPBS. For preparation of the lysates, cell pellets were resuspended in RIPA buffer of pH 8.0 containing 50 mM Tris, 100 mM NaCl, 0.1% w/v SDS, 1% v/v IGEPAL CO-520, 0.5% w/v deoxycholic acid, and 1 mM EDTA. Cells were incubated for 10 min on ice, centrifuged at 13′000 rpm for 15 min, and the soluble lysates were transferred into fresh Eppendorf tubes. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and measuring the absorbance at 562 nm using a multiplate reader (Spark, Tecan, Männedorf, Switzerland).

For preparation of cell lysates, the supernatants were collected following the co-culture experiment, and the cells were detached using Enzyme-Free Cell Dissociation Solution Hank’s Based (1 ×) (Merck KGaA, Darmstadt, Germany) and added to the supernatant. Cells were centrifuged at 800 rcf for 10 min and washed twice with 5mL DPBS. For preparation of the lysates, cell pellets were resuspended in RIPA buffer of pH 8.0 containing 50 mM Tris, 100 mM NaCl, 0.1% w/v SDS, 1% v/v IGEPAL CO-520, 0.5% w/v deoxycholic acid, and 1 mM EDTA. Cells were incubated for 10 min on ice, centrifuged at 13′000 rpm for 15 min, and the soluble lysates were transferred into fresh Eppendorf tubes. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and measuring the absorbance at 562 nm using a multiplate reader (Spark, Tecan, Männedorf, Switzerland).