Splenocytes were prepared from the spleens of 3 weeks old SPF chickens (Rhode Island Red, Roslin) via density gradient centrifugation by using Histopaque 1083 (Merck Life Science) according to the manufacturer’s protocol. Briefly, spleens were mashed through 100 µm cell strainer (Merck Life Science) and suspended in complete Roswell Park Memorial Institute 1640 (RPMI) medium containing 10% FBS and 0.1% penicillin and streptomycin. This was followed by layering with Histopaque 1083 (Merck Life Science) and centrifuging at 400× g for 30 min. The splenocytes were then harvested from the ‘buffy coat’ interface in the density gradient and resuspended in a fresh complete RPMI media. About 2 × 106 cells were plated on each well of 24 well plate and treated with 10 μg of rH9HA or 14 μg of rH9HA-Dec205/CD11c scFv (containing 10 μg rH9HA according to the molecular weight) or 10 μg of scFv or 10 μg of control scFv (anti-H9HA). All cells were stimulated for 5, 22, and 30 h in vitro at 41 °C.
Histopaque 1083
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Sao Tome and Principe, Israel
Histopaque-1083 is a density gradient medium used for the isolation of mononuclear cells from whole blood. It is a sterile, endotoxin-tested solution composed of polysucrose and sodium diatrizoate, adjusted to a density of 1.083 g/mL.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (12 mentions)
Streptomycin, by Thermo Fisher Scientific (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Dnase 1, by Merck Group (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Facsaria 2, by BD (7 mentions)
Histopaque 1119, by Merck Group (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
Hyaluronidase, by Merck Group (5 mentions)
Rpmi 1640 medium, by Merck Group (5 mentions)
Collagenase d, by Merck Group (5 mentions)
Egm 2mv, by Lonza (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (4 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (4 mentions)
Histopaque 1077, by Merck Group (4 mentions)
Facsdiva software, by BD (4 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (4 mentions)
278 protocols using histopaque 1083
1
Chicken Splenocyte Isolation and Stimulation
2
Isolation and Co-culture of Leukocytes and DH82 Cells
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The mononuclear cell fraction was isolated from heparinized blood with Histopaque 1083 (Sigma-Aldrich, USA) and used to inoculate a monolayer of DH82 cells (ATCC no. CRL-10389) according to Aguiar et al. (2008). Briefly, leukocytes were isolated by overlaying the buffy coat on Histopaque 1083 (Sigma-Aldrich, USA), and the interface containing the leukocyte fraction was collected and resuspended in 5 ml of Dulbecco's Modified Eagle's medium (Sigma-Aldrich, USA) supplemented with 10% heat-inactivated bovine calf serum (Hyclone Laboratories, USA). The leukocyte suspension was transferred to a 25 cm2 flask culture and incubated at 37℃ in a 5% CO2 atmosphere. After 24 hrs, 2 ml of fresh culture medium was added and the cells harvested 24 hrs later and added to a monolayer of uninfected DH82 cells (provided by Marcelo B. Labruna, University of São Paulo) in a 25 cm2 flask. The culture was maintained under the same conditions as above, except that the bovine calf serum was reduced to 2.5%, and the culture medium was partially (20%) replaced every 2~3 days. Cell cultures were monitored once a week for the presence of ehrlichial morulae by Diff-Quik staining according to the manufacturer's instructions (Laborclin Produtos para Laboratórios, Brazil) and ehrlichial DNA by nested PCR.
3
Lymphatic Vessel Cannulation in Calves
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Surgical cannulation of skin‐draining afferent lymphatic vessels was performed in seven calves at 3–6 months old, according to a protocol described in detail elsewhere.24 Briefly, prescapular LNs were excised under general anaesthesia and the calves were allowed to recover. LNs were collected into ice‐cold PBS, processed to release cells and mononuclear cells were obtained by density gradient centrifugation using Histopaque 1083 (Sigma Aldrich, Dorset, UK). After 8 weeks, following re‐anastomosis of the small afferent lymphatic vessels to the larger efferent lymphatic vessel, a sterile cannula was inserted into the ‘pseudo‐afferent’ lymphatic vessel. Lymph was collected into a tissue‐culture flask, secured to the side of the calf's head using a head‐collar and containing 3 g of sodium benzylpenicillin (Schering‐Plough, Kenilworth, NJ) dissolved in 5000 i.u/ml heparin (GP Pharmaceuticals, Barcelona, Spain). Flasks were changed twice daily, or as required, for up to 28 days post‐cannulation. Whole lymph was centrifuged to pellet the cells. Samples of PB were taken during the cannulation period by jugular venepuncture using 10‐ml vacutainers containing sodium heparin (10 U/ml), and peripheral blood mononuclear cells were subsequently separated by density gradient centrifugation using Histopaque 1083 (Sigma Aldrich).
4
Chicken Splenocyte Stimulation Assay
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Splenocytes were prepared from the spleens of 3-week-old SPF chickens (Rhode Island Red, Roslin) via density gradient centrifugation by using Histopaque 1083 (Merck Life Science) according to the manufacturer’s instructions. About 2 × 106 cells were plated on each well of a 24-well plate suspended in 300 μl of complete Roswell Park Memorial Institute 1640 (RPMI) medium containing 10% FBS and 0.1% penicillin and streptomycin. Cells were treated with 10 μg of rH9HA or 14 μg of rH9HA-CD83 scFv (containing 10 μg rH9HA according to the molecular weight) or 10 μg of CD83 scFv or 10 μg of control scFv (anti-H9HA). All cells were stimulated for 5, 22, and 30 h in vitro at 41 °C.
5
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.
6
Isolation of Tumor-Infiltrating DCs and T Cells
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MC38 tumor cell line was s.c. injected into mouse flanks (5 × 105 each side), and tumors were excised from euthanized mice without skin, lymph nodes, or connective tissues 10 days after the injection. Minced tumors were digested with Liberase TM (100 μg/mL) (Roche), Collagenase XI (250 μg/mL), Hyaluronidase (1 mg/mL), and DNase I (200 μg/mL) (Sigma-Aldrich) for 30 minutes at 37°C (41 (link)). The reaction was stopped by the addition of EDTA (20 mM final concentration). A single-cell suspension was prepared by sieving the digested tissue through a 70 μm nylon strainer (BD Biosciences). DCs and T cells were enriched by double-gradient centrifugation at 455g for 20 minutes at room temperature using 14.5% Nycodenz (Accurate Chemical) (low density for DCs) and Histopaque 1083 (MilliporeSigma) (high density for T cells). Cells were collected from each interface and then washed with PBS containing 0.5% BSA (Sigma-Aldrich) and 2 mM EDTA (Sigma-Aldrich).
7
Tissue-Engineered Vascular Graft Implantation
8
Murine Vena Cava Graft Transplantation
9
Splenic Immune Cell Isolation and Stimulation
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Spleen samples were harvested from mice on experimental diets after 20 weeks at the day of killing as previously described, with minor modification (22) . Erythrocytes were removed with Histopaque 1083 (MilliporeSigma) in a 15 ml conical tube and centrifuged at 300 g at 25°C for 30 min with deacceleration turned off. Splenocytes were then carefully removed from the opaque interface and washed two times with cold Roswell Park Memorial Institute (RPMI) media. Splenocytes were then plated at a concentration of 4•0 × 10 6 cells/well and treated with or without 500 ng/ml of LPS (Milli-poreSigma) for 20 h in RPMI media.
10
Isolation and purification of intrahepatic lymphocytes and spleen cells
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IHLs and spleen cells were prepared as described previously (33 (link)). Livers were perfused with 10 mL of PBS via the portal vein to remove circulating lymphocytes, and the liver tissue was pressed through a 70 μm cell strainer (Corning) with the plunger of a 1 mL syringe and digested with 10 mL of RPMI 1640 medium (MilliporeSigma), containing 0.02% (w/v) collagenase IV (MilliporeSigma) and 0.002% (w/v) DNase I (MilliporeSigma), for 40 minutes at 37°C. Cell suspension was collected after removing hepatocytes and connecting tissues by slow-speed centrifugation (3 minutes, 20g). The cells were washed with RPMI 1640 and then overlaid on Percoll/Histopaque solution consisting of 12% Percoll (GE Healthcare, now Cytiva) and 88% Histopaque-1083 (MilliporeSigma). After centrifugation for 20 minutes at 750g, the IHLs were isolated at the interface. The lymphomononuclear cells were washed twice with RPMI 1640 medium and used for further analysis. Spleen cells were isolated by pressing through a 70 μm cell strainer, washed 3 times with RPMI 1640 medium containing 5% FBS, and used for further analysis. For adoptive transfer, spleen cells were further filtered out with a 70 μm cell strainer and washed with HBSS (MilliporeSigma) twice.
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