Clspa

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Adipose tissue mononuclear cells (ATMCs) were isolated from intra-abdominal fat samples, which were first dissected then dissociated into a single cell suspension using a GentleMACS device (Miltenyi). Samples were digested in 2 ml RPMI medium (Gibco) supplemented with 20 U/ml CLSPA (Worthington) for every 4 g fat, over 1 h at 37 °C. The resulting cell suspension was washed in RPMI, and the fat layer that formed after centrifugation was decanted before the cell pellet was resuspended and filtered through a 40-µm nylon mesh. The cell suspension was then separated by Ficoll density gradient centrifugation. Isolated PBMCs and ATMCs were stored in cryopreservation medium at 10⁶ cells/100 µl [70% FCS (Gibco), 20% RPMI, 10% DMSO] in liquid nitrogen.

Human BM and blood samples were obtained from systemically healthy individuals who did not receive immunomodulatory drugs or suffer from diseases known to influence the immune system, including autoimmune diseases and cancer. Hip replacement surgery was performed and bone from the femur shaft was harvested. A biopsy of substantia spongiosa osseum, which would otherwise have been discarded, was used to isolate BMMCs. BM biopsies were fragmented, washed once with complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin; Invitrogen) and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/mL in complete RPMI medium) for 1 h at 37°C. BM biopsies were then centrifuged and BMMCs purified by density gradient centrifugation (Ficoll‐Hypaque). Experiments were performed using BMMCs and not with all BM cells in order to obtain cleaner populations and thus to improve the quality of the results. qPCR experiments comparing the expression of effector/memory cell survival factors in BMMCs and in BM cells showed similar results (data not shown). PBMCs were used for pilot and stimulation control experiments. Purification of PBMCs from heparinized blood was also performed by density gradient centrifugation.

Paired BM and blood samples were obtained from 55 systemically healthy individuals (20 females, 25 males, age range 36–89 years, mean age 64 ± 12.1 years) who did not receive immunomodulatory drugs or suffer from diseases known to influence the immune system, including autoimmune diseases and cancer. Bone from the femur shaft was harvested during hip replacement surgery, and a biopsy of substantia spongiosa osseum, which would otherwise have been discarded, was used to isolate BMMCs. BM biopsies were fragmented, washed once with complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin; Invitrogen), and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/mL in complete RPMI medium) for 1 h at 37°C. BM biopsies were then centrifuged and BMMCs were purified by density gradient centrifugation. Fresh BMMCs and PBMCs were used for the experiments.

Egg chambers were dissected in live imaging medium, and then incubated with collagenase (1,000 Units ml⁻¹ CLSPA; Worthington Biochemical Corp.) dissolved in the final volume of 100 μl culturing medium for 20 min before being mounted for imaging. For the chemical inhibition of Rho1 activity, dissected egg chambers were incubated with two Rho inhibitors: C3 exoenzyme61 (link) (Enzo Life Sciences) at 10 μg μl⁻¹ or Rhosin62 (link) (Merk) at 250 μM for 20 min before being mounted for imaging.

Samples were obtained from systemically healthy individuals who do not suffer from diseases known to affect the immune system. All samples were obtained from people undergoing elective surgeries because of osteoarthrosis. The donors comprised of 95 individuals aged between 39 and 87 (mean age: 67.45 ± 10.95, mean BMI: 27.9 ± 5.03, sex: 50 F, 46 M). The number of samples used in individual experiments are given in the figures and legends.
For the isolation of bone marrow mononuclear cells (BMMCs), a fragment of substantia spongiosa osseum, which would otherwise be discarded was collected during routine hip replacement surgery. The bone was further fragmented and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/ml) in complete RPMI medium (RPMI 1640; Corning supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin; Sigma) for 1 h at 37 °C. BMMCs were extracted using a filtered tube centrifugation step, and then purified using density gradient centrifugation (Lymphoprep®; Stemcell technologies). Heparinised blood from the same donors was collected, and peripheral blood mononuclear cells (PBMCs) were also purified by density gradient centrifugation.

Hip replacement surgery was performed and bone from the femur shaft was harvested. A biopsy of substantia spongiosa osseum, which would otherwise have been discarded, was used to isolate BM mononuclear cells (BMMCs). BM biopsies were fragmented, washed once with complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin; Invitrogen) and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/ml in complete RPMI medium) for 1 h at 37°C. BM biopsies were then centrifuged and BMMCs purified by density gradient centrifugation (Ficoll-Hypaque). Purification of PB mononuclear cells (PBMCs) from heparinized blood was also performed by density gradient centrifugation.