Heparinized anti-coagulated peripheral blood was collected from the severe sepsis patients. Granulocytes were isolated following the method described previously [13 (link)]. In brief, the whole blood cells were separated by lymphocyte separation medium (density of 1.077 g/ml, Histopaque®-1077, Sigma-Aldrich, USA) and Histopaque®-1119 (density of 1.119 g/ml, Histopaque®-1119, Sigma-Aldrich, USA), followed by centrifuging at 300×g for 5 min, and 800×g for 20 min. Granulocytes were collected from the lower layer, followed by washing with PBS and then were resuspend by RPMI 1640 medium (Sigma-Aldrich, USA) with 10 mM HEPES, 2 mM glutamine, 10% heat-inactivated fetal bovine serum, and 50 mM 2-mercaptoethanol. Then, the granulocytes were further purified by discontinuous Percoll (Amersham Biosciences, Sweden) centrifugation. Monocytes were collected from the upper layer (Histopaque®-1077 layer) and further purified by CD14 antibody-conjugated microbeads (Miltenyi Biotec, Germany). The cells were cultured with RPMI 1640 medium. The recovery of cells was checked by trypan blue staining.
Histopaque 1119
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, France, Sao Tome and Principe
Histopaque 1119 is a laboratory reagent used for the separation and isolation of mononuclear cells from whole blood. It is a sterile, endotoxin-tested solution of a polysucrose and sodium diatrizoate, adjusted to a density of 1.119 g/mL.
Automatically generated - may contain errors
Lab products found in correlation
Histopaque 1077, by Merck Group (126 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (16 mentions)
Rpmi 1640, by Thermo Fisher Scientific (11 mentions)
Liberase tl, by Roche (10 mentions)
Percoll, by GE Healthcare (8 mentions)
Rpmi 1640 medium, by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Histopaque, by Merck Group (7 mentions)
Hanks balanced salt solution (hbss), by Merck Group (7 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Histopaque 1083, by Merck Group (6 mentions)
Hepes, by Thermo Fisher Scientific (6 mentions)
Trypan blue, by Merck Group (5 mentions)
Dnase, by Roche (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Celltrace violet, by Thermo Fisher Scientific (5 mentions)
Hepes, by Merck Group (4 mentions)
Dnase 1, by Roche (4 mentions)
267 protocols using histopaque 1119
1
Granulocyte Isolation from Sepsis Patients
2
Isolation of Neutrophils from Whole Blood
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Neutrophils were collected from the whole blood of healthy volunteers according to the standard procedure, which was approved by the Research Ethics Committee, Guangdong General Hospital (Guangzhou, China).
First, 3 mL of Histopaque-1119 (Sigma Chemical Co., St. Louis, Mo.) was placed in a 15 mL conical centrifugation tube and 3 mL of Histopaque-1077 (Sigma Chemical Co., St. Louis, Mo.) was layered onto the Histopaque-1119. Fresh whole blood of 6 mL was anticoagulated with heparin, layered onto the density gradient, and then centrifuged at 700 g for 30 min at room temperature. Neutrophils were collected from the interface of Histopaque-1077 and Histopaque-1119 and into a new 15 mL conical centrifugation tube, and then washed with 10 mL phosphate buffer solution (PBS, Gibco, Grand Island, NY). After 10 min centrifugation at 400 ×g, the supernatant was discarded and 3 mL erythrocyte lysing buffer (ACK, Gibco, Grand Island, NY) was added into the tubes and gently shaken for 10 min. Then, the tubes were centrifuged for 10 min at 400 ×g and the supernatant was removed. The resulting neutrophils were re-suspended in PBS and assayed for cell viability using trypan blue exclusion. The obtained preparations were >95% pure and viable. All procedures were performed at room temperature.
First, 3 mL of Histopaque-1119 (Sigma Chemical Co., St. Louis, Mo.) was placed in a 15 mL conical centrifugation tube and 3 mL of Histopaque-1077 (Sigma Chemical Co., St. Louis, Mo.) was layered onto the Histopaque-1119. Fresh whole blood of 6 mL was anticoagulated with heparin, layered onto the density gradient, and then centrifuged at 700 g for 30 min at room temperature. Neutrophils were collected from the interface of Histopaque-1077 and Histopaque-1119 and into a new 15 mL conical centrifugation tube, and then washed with 10 mL phosphate buffer solution (PBS, Gibco, Grand Island, NY). After 10 min centrifugation at 400 ×g, the supernatant was discarded and 3 mL erythrocyte lysing buffer (ACK, Gibco, Grand Island, NY) was added into the tubes and gently shaken for 10 min. Then, the tubes were centrifuged for 10 min at 400 ×g and the supernatant was removed. The resulting neutrophils were re-suspended in PBS and assayed for cell viability using trypan blue exclusion. The obtained preparations were >95% pure and viable. All procedures were performed at room temperature.
3
Isolation and Characterization of Murine Neutrophils
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ICR mice aged 6 weeks were sacrificed by cervical dislocation at the time of neutrophils harvest. Tibias and femurs were removed and stripped of their muscles. The BM was flushed using PBS, and cell aggregates were disrupted via filtration through 70-μm cell strainer (BD Bioscience) and washed with PBS. Cell suspension was layered in a ratio of 1 to 3 on top of Histopaque 1077 (Sigma Aldrich), after centrifugation, precipitate was resuspended the with PBS. The cell suspension was layered in a ratio of 1 to 2 on top of Histopaque 1119 (Sigma Aldrich), after centrifugation, neutrophils were recovered on the top of Histopaque 1119. Neutrophils were washed with PBS and then resuspended in RPMI Medium 1640. The purity of neutrophils was determined by immunofluorescence staining for Ly6G (almost 100% cells were positive for Ly6G). Neutrophil viability was analyzed using Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) according to the manufacturer’s procedure.
4
Neutrophil Isolation from Murine Bone Marrow
5
Neutrophil Isolation from Mouse Bone Marrow
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Neutrophils were isolated from mouse bone marrow using Histopaque-based density gradient centrifugation [36 (link)]. Mouse bone marrow cells were layered over Histopaque 1077 (Sigma, MO, USA) and Histopaque 1119 (Sigma, MO, USA), and centrifuged for 30 min at 872 g. Neutrophils were collected from the Histopaque 1077 and Histopaque 1119 interface and washed with RPMI 1640 supplemented with 10% FBS and centrifuged for 7 min at 427 g.
6
Immune Cell Analysis of Pancreatic Tumors
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Samples were processed and analyzed as previously described (57 (link)). Pancreatic tumors were processed 24 hours after completion of the first cycle of therapy: 5 doses of TH-302 administered once daily and 2 doses of αVEGFR administered every 4 days. The pellet from the Histopaque-1119 (MilliporeSigma, 11191) separation was processed for endothelial cell analysis. CD8+ T cells were isolated using a magnetic-activated cell sorting CD8a+ T Cell Isolation Kit (Miltenyi Biotec, 130-104-075), activated for 6 hours using the eBioscience Cell Stimulation Cocktail plus protein transport inhibitors (Thermo Fisher Scientific, 00-4975-93), and analyzed by flow cytometry as previously described (56 (link)). Flow data were collected on a 5-laser, 18-color BD LSR II and analyzed using FlowJo version v10.7. A representative illustration of the immune cell gating scheme is provided in Supplemental Figure 5 .
7
Isolation of Osteoprogenitor Cells
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For the isolation of osteoprogenitors, perinatal mice from Jun-inducible mice (both systemic and bone-specific) were euthanized. After removing the skin, their bones (tibia, fibula, femur, spine, rib cage, humerus, radius, ulna, and skull) were harvested, dissected with scissors, smashed with a pestle, and digested in α-minimal essential medium (Gibco) substituted with 2.2 mg of collagenase I/mL (Thermo Fisher Scientific, MA, USA) for up to three rounds of 25 min under constant agitation at 37°C in the cell incubator. Thereafter, debris and red blood cells were removed by gradient centrifugation with Histopaque-1119 (MilliporeSigma). After washing, cells were stained with primary antibodies for 45 min and with the secondary antibody for 20 min (Table S1 ). Cells were then resuspended in FACS buffer (PBS + 2% FCS + 1% penicillin/streptomycin + 1 mM EDTA + 25 mM HEPES) and sorted for different subsets of osteoprogenitors using the BD FACSAria III sorter (Becton Dickinson). To mark negative and positive gates, we used FMO (full-minus-one) controls. Populations were double-sorted to ensure purity. We used FlowJo (FlowJo LLC) in its newest version for the analysis.
8
Isolation and Culture of Pancreatic Islets
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Mice were killed by cervical dislocation, and islets were isolated essentially as described67 (link). In brief, pancreata were inflated with collagenase solution at 1 mg/ml (Collagenase NB 8 Broad Range, Nordmark, S1745602), resected and placed in a water bath at 37 °C for 10 min. After washing, islets were purified on a Histopaque-1119 (Merck, #11191) / Histopaque-1083 (Merck, #10831) gradient. Islets were cultured overnight (or for 48 h, as indicated) in RPMI-1640 medium containing 11 mM glucose (control) or 25 mM glucose (diabetic), plus foetal bovine serum (10% (v/v), penicillin (100 U/ml) and streptomycin (0.1 mg/ml) solution (all Thermo-Fischer-Scientific) at 37 °C, in a humidified atmosphere of 5%CO2/95% air. The glucose concentration of RPMI was chosen to reflect the randomly fed blood glucose of the animals.
9
Isolation and Characterization of Granulocytes from Heparinized Blood
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Briefly, 15 mL Histopaque 1119 (Merck, Darmstadt, Germany) was overlaid with 15 mL Histopaque 1077 solution. Then, 20 mL of heparinized blood (100 I.U. sodium heparin/mL, Roth, Karlsruhe, Germany) was carefully overlaid, followed by centrifugation at 630× g without braking at 18 °C for 25 min. The mononuclear cell fraction located between the plasma and Histopaque 1077, and the second layer containing granulocytes were collected. After washing, and resuspension of cell fractions in phosphate-buffered saline (PBS without Ca++/Mg++; glucose (1 g/L; 5.5 mM), Merck, Darmstadt, Germany), cell number was determined using an ADVIA-120 blood cell counter (Siemens, München, Germany). One part of the granulocyte suspension was used for degranulation experiments (see Section 2.1.3 ), another part was used for direct measurement of enzyme activities.
10
Dual Density Neutrophil Isolation
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As a second neutrophil isolation method and for comparison of neutrophil function, neutrophils were isolated using a dual density–separation method with a combination of Histopaque®-1119 (Sigma-Aldrich, Darmstadt, Germany) and a second gradient density separation using PercollTM PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), as described before [25 (link)].
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