Dulbecco s phosphate buffered saline dpbs

Peripheral blood mononuclear cells (PBMCs) were isolated using Percoll (GE Healthcare) density gradient centrifugation. Human peripheral blood was collected into 3.8% sodium citrate and centrifuged for 20 min at 350g. Following aspiration of plasma, leukocytes were separated from erythrocytes by 6% dextran sedimentation for 30 min and washed in 0.9% saline. PBMCs were then isolated by a discontinuous Percoll gradient of 81, 70, and 55% with the cells resuspended in 55% Percoll (Sigma-Aldrich) and carefully layered onto the 70% layer. The gradient was then centrifuged for 20 min at 700g (20°C, brake 0, acceleration 0), and PBMCs were collected using a Pastette from the 55/70 interface. These cells were then washed twice in cation-free 1× Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific). Monocytes were isolated from the PBMCs by MACS cell separation using a pan-monocyte, negative selection magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec, #130-096-537).

Skin MCs (~10⁶ cells/well) were kept in minimal medium consisting of Basal
Iscove medium (with stable glutamine; Biochrom, Berlin, Germany), supplemented with 0.5%
BSA (Serva, Heidelberg, Germany). MCs were left untreated or stimulated with SCF
(Peprotech, Rocky Hill, CT, USA) (at 10 nM) or treated with staurosporine (Enzo
Life Sciences, Lörrach, Germany) (at 2 μM) for the indicated
times. After incubation, cells were washed with 1×Dulbecco's phosphate-buffered
saline (DPBS) (Thermo Fisher Science, Berlin, Germany) and processed for downstream
applications (as described below).

The colon and part of the cecum were opened lengthwise and cut into 2- to 4-cm pieces, collected in 15 ml of ice-cold 1× RPMI 1640 buffer (Gibco) in a 50-ml Falcon tube, and cleaned with 20 ml of ice-cold 1× Dulbecco’s phosphate-buffered saline (DPBS; Gibco) in another 50-ml Falcon tube. The tissue was then placed into 15-ml conical centrifuge tubes filled with 10 ml of ice-cold dissociation reagent 1 (30 mM EDTA, 1.5 mM dithiothreitol [DTT], diluted into 1× DPBS) and placed on ice for 20 min. Tissues were then placed into a 15-ml conical centrifuge tube filled with 6 ml of warm (37°C) dissociation reagent 2 (30 mM EDTA, diluted into 1× DPBS) and incubated for 10 min at 37°C. After this incubation, tubes were shaken vigorously for 30 s to detach the epithelium from the basement membrane, for a total of about 80 to 90 shake cycles. Remnant intestinal tissue was removed, and the pellet cell solution was centrifuged at 800 × g for 5 min at 4°C. The supernatant was removed, and the cell pellet was resuspended in 1 ml of Tri reagent (Molecular Research Center) for subsequent RNA extraction or in radioimmunoprecipitation assay (RIPA) buffer for metabolism analysis.

The colon and part of cecum were opened lengthwise and cut into 2 to 4 cm pieces, collected in 15 mL of ice-cold 1× RPMI 1640 buffer (Gibco, Waltham, MA, USA) in a 50 mL Falcon tube and cleaned with 20 mL of ice-cold 1× Dulbecco’s phosphate-buffered saline (DPBS; Gibco) in another 50 mL Falcon tube. The tissue was then placed into 15 mL conical centrifuge tubes filled with 10 mL of ice-cold dissociation reagent 1 (30 mM EDTA, 1.5 mM dithiothreitol (DTT), diluted into 1× DPBS) and placed on ice for 20 min. Tissues were then placed into a 15 mL conical centrifuge tube filled with 6 mL of warm (37 °C) dissociation reagent 2 (30 mM EDTA, diluted into 1× DPBS) and incubated for 10 min at 37 °C. After this incubation, tubes were shaken vigorously for 30 s to detach the epithelium from basement membrane, for a total of about 80 to 90 shake cycles. Remnant intestinal tissue was removed, and pellet cell solution was centrifuged at 800× g for 5 min at 4 °C. Supernatant was removed, and the cell pellet was re-suspended in radioimmunoprecipitation assay (RIPA) buffer for metabolism analysis.

FACS-isolated and expanded FAPs were resuspended in 50 µL of ice-cold 1X Dulbecco's Phosphate-Buffered Saline (DPBS; Gibco, Billings, MT) and injected into the gastrocnemius muscle of SCID mice as previously described (Lees-Shepard et al., 2018 (link)). In most cases, both gastrocnemius muscles of an individual mouse were injected. For a given treatment group, variation in HO volume within and between mice was not statistically different, and each injection was treated as an independent event for statistical analysis. The gastrocnemius muscle was pinch-injured the day of transplantation using 3500 – 3700 grams of force applied with a Randall Selitto Paw Pressure Test Apparatus (IITC Life Science, Woodland Hills, CA). Treated SCID host mice received a single subcutaneous dose of ActA-mAb (10 mg/kg) on the day of injury, or daily IP injections of 1.47 mg/kg palovarotene or vehicle, beginning 3 days prior to injury and transplantation.

Blood samples were processed according to a previously described protocol (21 (link)). The cells (from 1 ml blood) were mixed with 10 ml fixation mixture (FM) in 50-ml plastic tubes and incubated for 10 min at 4°C. After centrifugation at 800 x g for 5 min at room temperature (RT), red cells were lysed by adding 10 ml Milli-Q water at RT for 20 min, without agitation. After two washes with 1X DPBS, cells were counted and stored at −80°C in FM at a final concentration of 15 × 10⁶ cells/ml and distributed into aliquots containing 3 × 10⁶ cells. FM used to fix and store the cells was prepared the day before the experiments and conserved at 4°C. The 5% formaldehyde FM solution was prepared from 36% paraformaldehyde (VWR BDH Prolabo, Fontenay-sous-Bois) and contained 18.5% glycerol (Sigma-Aldrich, Lyon, France) in 1X-Dulbecco's phosphate buffered saline (DPBS), without CaCl₂ or MgCl₂, pH 7.4 (Gibco by life Technologies, Villebon-Sur-Yvette, France). This solution allowed freezing and recovery of all blood leukocytes, especially polymorphonuclear cells, which are highly labile and cryopreservation-sensitive. Healthy and HIV-infected samples used for the multi-tube 72-marker experiment were cryopreserved for a maximum of 12 days.