Histopaque 1077

Human heparinized BM aspirate (10 ml) was collected from healthy and SLE patients and subjected to density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich). Briefly, blood was diluted 1:3 PBS and carefully layered over Histopaque medium. Tubes were centrifuged at 500 g for 30 min (no break) at room temperature. BM mononuclear cells were isolated using Histopaque-1077 (Sigma-Aldrich). BM mononuclear cells were washed, and erythrocytes were lysed with RBC buffer (420301, BioLegend). CD34⁺ cells were isolated using EasySep™ Human CD34 Positive Selection Kit II (18056, StemCell Technologies). Purity was tested and was >95%.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by utilizing density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich). To obtain lung leukocytes from IPF or control lungs, lung tissue was minced using dissection scissors to an approximate size of 2 mm³. Minced tissue was digested in RPMI media containing 10% FBS, 240 U/mL Collagenase IV (Sigma-Aldrich), and 4 μg/mL DNase I (Worthington Biochemical Corporation, Lakewood, NJ, USA) at 37°C for 90 min. Digested tissue was filtered through nylon mesh, and mononuclear cells were enriched by gradient centrifugation using Histopaque-1077 (Sigma-Aldrich). LLN cells were extracted from multiple hilar lymph nodes by mechanical dissociation. Isolated cells from the blood, lung, and LLN from IPF and control tissues were cryopreserved until analysis.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat cell packages donated for research and no longer usable for humans, obtained from the Blood Bank of the General Hospital of México “Eduardo Liceaga”. PBMCs were isolated from blood by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA) at 325×g for 20 min at 20 °C. Briefly, after 1:1 dilution with PBS pH 7.2 (Gibco Life Technologies, Waltham, MA, USA), blood samples are slowly and steadily placed on top of Histopaque-1077 (Sigma-Aldrich), in centrifuge tubes and centrifuged at 325×g for 10 min. The cells were carefully harvested from the interphase, washed with cold PBS pH 7.2 buffer, and incubated for 15 min in lytic solution to lyse residual erythrocytes. The PBMCs obtained were washed with PBS pH 7.2 and counted in a hemocytometer (Millipore, Burlington, MA, USA) using the trypan blue dye exclusion method.
The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Universidad Nacional Autónoma de México (reference number FM/DI/038/2021).

Whole blood samples were obtained in sodium heparin treated tubes from the NIH blood bank (Bethesda, MD). The study was reviewed and approved by Institutional Review Board and Food and Drug Administration (IRB-FDA) and Research Involving Human Subjects Committee (RIHSC), IRB-FDA, RIHSC Protocol #12-050R (Silver spring, MD). All donors were in good health and blood samples were negative for blood-borne pathogens as detected by standard blood bank assays. The peripheral blood mononuclear cells (PBMCs) were enriched by Histopaque 1077 (Sigma, St. Louis, MO) according to standard laboratory procedures. Briefly, 25ml of whole blood was loaded on top of 10ml of Histopaque 1077 (Sigma, St. Louis). The mix was centrifuged at 400g with brakes off and at room temperature for 30min. Interfacial cells were gently collected, washed with Hank’s balanced salt solution (HBSS) (Gibco, Green Island, NY) and resuspended in M199 medium (Sigma) with 10% heat inactivated AB serum (Sigma) supplemented with penicillin and streptomycin (Gibco). Cells were counted and adjusted to 5 x 10⁶ cells / ml.

Five milliliters of anticoagulated blood were layered onto 3 mL of Histopaque-1077 (Sigma-Aldrich) and lymphocytes were isolated from the plasma/Histopaque-1077 interface after centrifugation (10 min, 400×rcf) at room temperature. Cells were washed by adding PBS until a volume of 10 mL was reached and resuspended in restimulation medium (Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% FBS, l-glutamine and antibiotics) after centrifugation (10 min, 250×rcf). Splenocytes were isolated by forcing 1cm³ of splenic tissue over a 70 μm mesh cell strainer (Corning, Oneonta, NY, USA). Red blood cells were lysed by resuspension of cells in 5 mL of NH₄Cl solution before resuspending immune cells in 5 mL of restimulation medium.

Mouse islets were isolated following an injection of 0.6 mg/mL Collagenase P (Roche) into the pancreatic bile duct. Partially dissociated tissue was fractionated using a Histopaque-1077 (Sigma) gradient followed by hand-picking of islets. Islets were isolated by injection of 0.6 mg/mL Collagenase P (Roche) into the pancreatic bile duct, and partially dissociated tissue was fractionated using a Histopaque-1077 (Sigma) gradient followed by hand-picking of islets. Islet isolations were performed by the Vanderbilt Islet Procurement and Analysis Core.

As described previously [7 (link),30 (link),41 (link),63 (link)], mLNs were harvested and grinded by slid glass. Through a 100 nm nylon mesh, the aggregated cells were removed and the mLN cell were digested with digestion buffer which contained with DNase I (Sigma-Aldrich) and collagenase type IV (Sigma-Aldrich). After washing with PBS, the cells were re-suspended with 5 mL of histopaque-1077 (Sigma-Aldrich) and upper layered with 5 mL of fresh histopaque-1077, and followed density cut by centrifugation at 2000× g for 10 min. The cells heavier than 1.077 were harvested as the leukocytes and suspended PBS.