Hanks balanced salt solution (hbss)

Epididymal fat was dissected and collected into Hanks Basic Salt Solution (HBSS; all chemicals from Sigma-Aldrich). For each pre-adipocyte preparation, 7 g fat was chopped, placed into 30 ml digestion solution (45 mg collagenase type II and 675 mg BSA in HBSS), shaken at 180 rev/min for 40–50 min at 37°C, strained through a 100 μm mesh (BD Falcon, Tewksbury, MA, USA) and placed on ice for 20 min. The upper layer containing mature adipocytes was then removed. The top two-thirds was mixed with growth medium (high glucose DMEM supplemented with 10% newborn calf serum, 1% penicillin-streptomycin and 200 μmol/l l-glutamine). The stromal–vascular fraction was centrifuged and re-suspended in growth medium. Following a second centrifugation, the pellets were re-suspended in 5 ml erythrocyte lysis buffer (150 mmol/l ammonium chloride, 10 mmol/l potassium bicarbonate, 0.1 mmol/l EDTA, pH 8.0) for 5 min. Cells were pelleted, re-suspended in growth medium, counted and plated at a density of 1 × 10⁴ per cm² into 75 ml flasks. The culture medium was supplemented with an adipogenic cocktail (30 μmol/l insulin [Actrapid, Novo Nordisk, Gatwick, UK], 150 μmol/l sodium ascorbate) daily for the first 3 days and then every other day until harvesting on day 11.

Pancreatic tissues were carefully removed and immediately resuspended in 3 ml of Hank’s balanced salt solution (HBSS; Wako, Osaka) on ice. The tissues were cut into small pieces and digested in HBSS containing 2.5 mg/ml collagenase P and 0.1 mg/ml DNase I for 30 min at 37 °C. After incubation, the cells were washed with cold sterile phosphate-buffered saline (PBS) and collected by centrifugation at 1500 r.p.m. for 10 min. The cell pellets were resuspended in a cold HBSS medium, filtered through a 100-µm strainer (BD Falcon, no. 352360), and further collected by centrifugation at 1500 r.p.m. for 10 min. The cells were counted with a hemocytometer, and a Countess II FL automated cell counter (Invitrogen) was used. For edema analysis, whole pancreas samples were weighed and dried at 95 °C for 48 h. Edema was calculated following desiccation and is expressed as a ratio of the wet weight (wet weight  − dry weight/wet weight × 100).

Skin-infiltrating leukocytes were isolated from the back skin or left ear skin of IMQ-induces psoriasis mice (28 (link)). After 7 days of IMQ application, the 1 cm² area from the mouse back skin and left ear were cut off, removed with remaining IMQ cream by HBSS (Gibco) wash and separated with epidermis and dermis using curved forceps. Separated epidermis and dermis were digested with Dispase II solution (5 mg/ml, Sigma Aldrich) at 37°C. After first digestion, the dermis was cut quickly into small pieces (< 0.5 mm) with curved scissors in Dermis Dissociation buffer (DMEM containing 1 mg/ml of collagenase P and 100 μg/ml of DNaseI (Hyclone, Roche) and incubated at 37°C for 1 hr. The fully digested dermal suspension was filtered through a 40-μm cell strainer (Falcon) and rinsed with DMEM medium containing 10% FBS (Hyclone). Isolated dermal-infiltrating leukocytes were collected by centrifugation. The epidermis, after first digestion, was cut quickly into small pieces (< 2 mm) with curved scissors and transferred to the 0.05% TE (Trypsin-EDTA, Hyclone) buffer and incubated at 37°C for 5 min. After incubation, trypsin neutralizing solution (5% FBS diluted with HBSS) was added to the fully digested epidermis, and the epidermal suspension was filtered through a 40-μm cell strainer (Falcon). Isolated epidermal-infiltrating leukocytes were collected by centrifugation.

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from a buffycoat after informed consent (Blood Transfusion Centre, Ghent), using a Lymphoprep (Axis-Shield, Dundee, Scotland) gradient. Fifty ml of the buffycoat was added to 250 ml Hank’s Balanced Salt Solution, without Ca²⁺ and Mg²⁺ (HBSS) (Invitrogen). Of this dilution, eight aliquots of 35 ml were each added to 15 ml Lymphoprep in a 50 ml Falcon tube. These mixtures were subsequently centrifuged at 500 x g for 20 min at room temperature. The inner whitish ring of PBMCs, present between the Lymphoprep and the plasma phase, was transferred to 25 ml HBSS and centrifuged at 450 × g for 10 min at room temperature. The supernatants was removed and the cell pellet was resuspended in 10 ml HBSS. All resuspended cells were pooled into a 50 ml Falcon tube and HBSS was added to a total volume of 50 ml. A small fraction of this cell solution was used to count the number of cells present, before it was centrifuged again at 350 × g for 10 min at room temperature.
The total number of cells was counted using a Sysmex KX-21 (Sysmex, Norderstedt, Germany). The cell pellet was resuspended in heat-inactivated foetal calf serum with 10% dimethyl sulfoxide (DMSO) to a concentration of 2 × 10⁷ cells/ml and divided in 1 ml aliquots before cryostorage them in liquid nitrogen.

Epididymal fat was dissected and collected into Hanks Basic Salt Solution (HBSS; all chemicals from Sigma-Aldrich). For each pre-adipocyte preparation, 7 g fat was chopped, placed into 30 ml digestion solution (45 mg collagenase type II and 675 mg BSA in HBSS), shaken at 180 rev/min for 40–50 min at 37°C, strained through a 100 μm mesh (BD Falcon, Tewksbury, MA, USA) and placed on ice for 20 min. The upper layer containing mature adipocytes was then removed. The top two-thirds was mixed with growth medium (high glucose DMEM supplemented with 10% newborn calf serum, 1% penicillin-streptomycin and 200 μmol/l l-glutamine). The stromal–vascular fraction was centrifuged and re-suspended in growth medium. Following a second centrifugation, the pellets were re-suspended in 5 ml erythrocyte lysis buffer (150 mmol/l ammonium chloride, 10 mmol/l potassium bicarbonate, 0.1 mmol/l EDTA, pH 8.0) for 5 min. Cells were pelleted, re-suspended in growth medium, counted and plated at a density of 1 × 10⁴ per cm² into 75 ml flasks. The culture medium was supplemented with an adipogenic cocktail (30 μmol/l insulin [Actrapid, Novo Nordisk, Gatwick, UK], 150 μmol/l sodium ascorbate) daily for the first 3 days and then every other day until harvesting on day 11.

Epididymal fat was dissected and collected into Hanks Basic Salt Solution (HBSS; all chemicals from Sigma-Aldrich). For each pre-adipocyte preparation, 7 g fat was chopped, placed into 30 ml digestion solution (45 mg collagenase type II and 675 mg BSA in HBSS), shaken at 180 rev/min for 40–50 min at 37°C, strained through a 100 μm mesh (BD Falcon, Tewksbury, MA, USA) and placed on ice for 20 min. The upper layer containing mature adipocytes was then removed. The top two-thirds was mixed with growth medium (high glucose DMEM supplemented with 10% newborn calf serum, 1% penicillin-streptomycin and 200 μmol/l l-glutamine). The stromal–vascular fraction was centrifuged and re-suspended in growth medium. Following a second centrifugation, the pellets were re-suspended in 5 ml erythrocyte lysis buffer (150 mmol/l ammonium chloride, 10 mmol/l potassium bicarbonate, 0.1 mmol/l EDTA, pH 8.0) for 5 min. Cells were pelleted, re-suspended in growth medium, counted and plated at a density of 1 × 10⁴ per cm² into 75 ml flasks. The culture medium was supplemented with an adipogenic cocktail (30 μmol/l insulin [Actrapid, Novo Nordisk, Gatwick, UK], 150 μmol/l sodium ascorbate) daily for the first 3 days and then every other day until harvesting on day 11.

Resected tumors were transported in Hank’s Balanced Salt Solution (HBSS, Life Technologies) on ice immediately after surgery. The tumor sample was subsequently divided into two pieces, and a small fragment was stored in liquid nitrogen for tissue staining. The remainder of the tumor was minced with scalpels into tiny cubes <0.5 mm³ and transferred into a 15 mL conical tube (BD Falcon) containing 8 mL pre-warmed HBSS, 1 mg/mL collagenase I and 0.5 mg/mL collagenase IV. Tumor pieces were digested on a Tube Revolver (Thermo) for 30 min at 37 °C. This suspension was then filtered using a 70 μm nylon mesh (BD Biosciences) and residual cell clumps were discarded, then the cell pellet was resuspended in red blood cell lysis buffer. Following a 5 min incubation at room temperature, samples were centrifuged to discard the supernatant and re-suspend the cell pellet in PBS with 0.04% FBS. Cell sorting was performed with a MoFloAstrios EQ (Beckman Coulter). Live cells were used for single-cell experiments after the dead cells were eliminated based on exclusion of 7-aminoactinomycin D (Life Technologies).