Accutase solution

Isolation of human adipose-tissue derived mesenchymal stromal cells was performed as it was described previously [39 (link)]. All procedures were conducted under sterile conditions. Briefly, tissue biopsies were washed extensively with Hanks’s Balanced Salt Solution (HBSS) complemented with 1% antibiotic-antimycotic solution. Next, tissues were sliced into small pieces using surgical scissors. Tissue fragments were digested with 1 mg/mL collagenase type I for 40 min at 37 °C and then centrifuged for 10 min at 1200× g. Obtained pellets of stromal vascular fraction were suspended in Dulbecco’s Modified Eagle’s Medium (DMEM) with Nutrient F-12 Ham supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antymycotic solution, and after that transferred to culture flasks. Primary cultures were maintained at 37 °C in an incubator with a humidified atmosphere of 5% CO₂. When cultures reached approximately 80%, the passage of cells was performed. In order to obtain a sufficient number of cells for the experiment, cultures were passaged three times. Passage was performed using Accutase^® solution (Biowest S.A.S., Nuaillé, France) according to the manufacturer’s instructions. Following harvesting, cells were washed with HBSS at 300× g for 4 min.

Cultures of RAW264.7 were maintained at 37 °C in incubator with a humidified atmosphere. RAW264.7 were propagated in DMEM containing 4500 mg/L of glucose, supplemented with 10% FBS and 1% antibiotic-antymycotic solution. Medium was changed every two days. The passage was performed when cells reached approximately 80% confluence. For cells detachment the Accutase^® solution (Biowest S.A.S., Nuaillé, France) was used. The removal of enzyme and cells washing was performed at 125× g for 4 min. Cells used for the experiment were established at 10th passage.

Cells were grown to confluence in 6-well plates, detached with Accutase solution (Biowest), washed with staining buffer (PBS buffer supplemented with 1% bovine serum albumin and 0.002% NaN₃) and incubated with or without 1 μl of calreticulin (D3E6) XP®Rabbit mAb #12238 for 2 h at 4°C. After the incubation, the cells were washed with staining buffer and incubated with 0.125 μg of fluorochrome-conjugated secondary antibody, PE Donkey anti-rabbit IgG (minimal x-reactivity) antibody (Biologend, clone Poly4064) or with 0.125 μg Alexa Fluor®647 Donkey anti-rabbit IgG (minimal x-reactivity) antibody (Biologend, Clone Poly4064), for 30 min at 4°C. Afterwards, cells were washed twice with staining buffer and fixed in 1% buffered formaldehyde. The samples were then immediately analyzed with a FACSCalibur II cytometer (BD Pharmingen). All analysis was carried out with Weasel 3.0.2 software (http://en.bio-soft.net/other/WEASEL.html).