Mouse neutrophils were isolated from the peritoneal cavities of LPS-induced endotoxin shock or CLP mice using the Histopaque-1077 solution (Sigma). Cell counts were carried out using Trypan Blue (Sigma). Human neutrophils were isolated from the peripheral blood of a healthy human donor using the Histopaque-1077 solution (Sigma) through a density gradient. The peripheral bloods were collected with written informed consent, which was approved by the Institutional Animal Care and Use Committee at Ajou University College of Medicine (Suwon, Korea). Mouse splenocytes were isolated from the spleen after CLP. Isolated spleens were ground using the Tissue Sieve System (Bellco Glass). Ground cells were filtrated by a 70-um Cell Strainer (BD Falcon), and blood cells were lysed by RBC lysis buffer. Splenocytes were gained through washing with PBS three times.
Histopaque 1077 solution
Manufactured by Merck Group
Sourced in Germany, United States
Histopaque-1077 is a sterile, endotoxin-tested solution designed for the isolation of mononuclear cells from human blood. It is a media prepared for the density gradient separation of blood components.
Automatically generated - may contain errors
Lab products found in correlation
Trypan blue, by Merck Group (2 mentions)
Red blood cell lysis buffer, by Merck Group (2 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
70 um cell strainer, by BD (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Vi cell cell counter, by Beckman Coulter (1 mentions)
Rnalater, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Dmem ham s f 12 medium, by Merck Group (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt dimethyl sulfoxide dmso, by Merck Group (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Fast powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin amphotericin b, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Ham s f 12, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Griess reagent, by Merck Group (1 mentions)
Oligo dt primer, by Bioneer (1 mentions)
12 protocols using histopaque 1077 solution
1
Isolation of Mouse and Human Neutrophils
2
Isolation of Canine Immune Cells
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Peripheral blood mononuclear cells were obtained through density gradient centrifugation (Histopaque®-1077 solution, Sigma-Aldrich, Germany). Dog peripheral blood was resuspended in PBS (1:1 v/v), overlaid on half of that total volume in Histopaque®-1077 solution and centrifuged 400 × g for 30 min at 18°C. Peripheral blood mononuclear cells were then harvested at the interface of PBS and Histopaque® and washed twice in cold PBS (300 × g, 10 min, 4°C). Whenever red blood cells were still visible in the pellet, a step of lysis was done by adding 5 ml of RBC Lysis Buffer (eBioscience, USA) for 5 min and stopping the reaction with 10 ml of PBS, followed by a centrifugation at 300 × g (4°C) for 10 min. The pellet was then resuspended in Flow Cytometry Staining Buffer (FCSB) (eBioscience), and the total volume was adjusted for 2 × 107 cells ml−1. Lymph node and bone marrow aspirates were centrifuged at 400 × g (4°C) for 5 and 15 min, respectively, and resuspended in FCSB with the total volume also adjusted for 2 × 107 cells ml−1. These samples were then kept on ice until antibody labeling.
3
Isolation and Preservation of Canine Immune Cells
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Dog peripheral blood was re-suspended in PBS (1:1 v/v), overlaid onto a 1:2 Histopaque®-1077 solution (Sigma-Aldrich, Germany) and centrifuged at 400 g for 30 min at 18°C. Mononuclear cells were harvested and washed in cold PBS (300 g, 10 min, 4°C), re-suspended in PBS, and the total volume adjusted to 2 × 107 cells ml−1. Lymph node and bone marrow aspirates were centrifuged at 400 g (4°C) for 5 and 15 min, respectively, and re-suspended in 100 μl, with the total volume also adjusted for 2 × 107 cells.ml−1. Then, 200 μl of peripheral blood mononuclear cells (PBMCs) and 100 μl of lymph node and bone marrow cell suspensions were centrifuged at 400 g (4°C) for 5 min, re-suspended in 600 μl of RLT Buffer (QIAGEN®) supplemented with β-mercaptoethanol and stored at −80°C until further use.
4
Isolation of Human Peripheral Blood Mononuclear Cells
5
Isolation of Pancreatic Islets from Rats
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Sprague-Dawley rats (male, 8 to 10 week of age; Samtako Bio Korea, Osan-si, Gyeonggi-do, Republic of Korea) were used as pancreatic islet donors. Briefly, rats were euthanized by cervical dislocation, pancreases were exposed, and 0.08% collagenase type P solution (Sigma-Aldrich, Saint Louis, MO, USA) was injected via the hepatic bile duct. Pancreases were then incubated for 18 min at 37°C. Pancreatic islets were isolated from exocrine cells by using a Histopaque-1077 solution (Sigma-Aldrich, Saint Louis, MO, USA) and cultured in RPMI 1640 growth medium (Mediatech, Manassas, VA, USA) containing 10% FBS and 1% penicillin-streptomycin antibiotics 100× (Thermo Fisher Scientific, Waltham, MA, USA) for 3 days to recover functionality before transplantation (74 ).
6
Isolation and Storage of PBMC
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Blood samples were drawn from the vein of the antecubital region following a 12-h fasting, and participants were asked to avoid intense physical exercise 72 h before sampling. All blood samples were drawn into 4-mL EDTA anticoagulant or serum separator tubes. Serum and plasma were routinely centrifuged at 1,500×g for 15 min. Plasma and serum were stored at −80°C for subsequent analysis. PBMC were immediately separated from anticoagulated peripheral blood by density gradient centrifugation with Histopaque®-1077 solution (Sigma-Aldrich, St. Louis, USA), as previously described [16 (link)],and with short modifications. For each sample, four 15-mL centrifuge tubes were used to layer 7 mL of blood and phosphate-buffered saline (PBS) (136 mM NaCl, 2.7 mM KCl, 7.8 mM Na2HPO4, 1.7 mM KH2PO4) onto 3 mL of Histopaque®-1077. The suspension was centrifuged for 40 min at 275×g at room temperature. After, the mononuclear cell layer was removed with manual pipetting and washed in PBS. Then, cell supernatants were discarded, and the PBMC pellets were dried out with lysing solution (150 mM NH4Cl, 10 mM NaHCO3, 1 mM EDTA) and centrifuged for 3 min at 300×g. Finally, samples were stored with RNAlater® (Sigma-Aldrich, St. Louis, USA) and frozen at −80°C for further analysis.
7
Monocyte-Cardiomyocyte Contractility Assay
8
Isolation of PBMCs from Whole Blood
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PBMCs were enriched by density gradient centrifugation using Histopaque-1077 solution (Sigma-Aldrich) according to the manufacturer’s instructions. Red blood cells were removed using a red blood cell lysis buffer (Sigma-Aldrich).
9
IPEC-J2 Cell Culture Optimisation
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Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/Ham’s F-12) medium (standard formulation containing arginine at 147.5 mg/L), Histopaque-1077® solution, penicillin/streptomycin/amphotericin B, glutamine, Trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), DNAse, Griess reagent, and phytohemagglutinin (PHA) were purchased from Sigma-Aldrich (St. Louis, MO, USA); foetal bovine serum (FBS) and TRI-reagent were obtained from ThermoFisher (Carlsbad, CA, USA); DMEM/Ham’s F-12 without arginine was obtained from US Biological Life Science (Salem, MA, USA). Oligo-dT primers were purchased from Bioneer (Daejeon, Korea). A high-capacity cDNA Reverse Transcription kit, and Fast Power-Up SYBR Green Master Mix were obtained from Applied Biosystems (Foster City, CA, USA), and primer sets from Eurofins Genomics (Ebersberg, Germany); IPEC-J2 were kindly provided by Dr. A. Baldi (Department of Veterinary Science for Health, Animal Production and Food Safety, University of Milan, Lodi, Italy).
10
Isolation and Enumeration of PBMCs
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