Rat brain blood vessels were isolated from the thalamus region after removing the choroid plexuses, following previously described methods (McNeill et al., 1999 (link); Placido et al., 2017 (link)). In brief, the extracted brain was homogenized in ice-cold Hanks balanced salt solution (HBSS, 137 mM Sodium chloride, 5 mM potassium chloride, 4.1 mM Sodium bicarbonate, 1.3 mM Calcium chloride, 0.5 mM Magnesium chloride, 0.4 mM Magnesium sulfate, 0.4 mM Potassium phosphate monobasic, 0.3 mM Sodium Phosphate dibasic, 5.5 mM Glucose, pH 7.4). After centrifugation at 2000 g for 10 min, the pellet was resuspended in ice-cold HBSS and layered over with 16% dextran solution (Sigma-Aldrich, St. Louis, MO, United States), followed by centrifugation at 4400 g for 15 min. The procedure was repeated twice to collect the top and middle layers containing the blood vessels, which were then filtered through a 20 μm nylon mesh. The vessels on the top of the nylon mesh were used for detection of RNA and protein expression levels of P-gp.
Dextran solution
Manufactured by Merck Group
Sourced in United States, Germany
Dextran solution is a laboratory product manufactured by Merck Group. It is a polysaccharide solution commonly used as a volume expander and plasma substitute in various applications. The core function of dextran solution is to maintain fluid balance and blood viscosity in in vitro and in vivo experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll hypaque density gradient solution, by GE Healthcare (4 mentions)
Dnase 1, by Roche (2 mentions)
Gelatin, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Santa Cruz Biotechnology (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
Fecl3 6h2o, by Carl Roth (1 mentions)
Fecl2 4h2o, by Merck Group (1 mentions)
Hank s balanced salt solution hbss, by Merck Group (1 mentions)
Rpmi culture medium, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Collagenase type xi, by Merck Group (1 mentions)
Cmrl 1066 medium, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Nissui Pharmaceutical (1 mentions)
Oligo dt and m mlv reverse transcriptase, by Promega (1 mentions)
Basic fibroblast growth factor (bfgf), by BioLegend (1 mentions)
L glutamine, by BioLegend (1 mentions)
Pdgf bb, by BioLegend (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Collagenase dispase, by Roche (1 mentions)
0 22 m membrane microfilters, by Corning (1 mentions)
18 protocols using dextran solution
1
Isolation of Rat Brain Blood Vessels
2
Isolation of Brain Microvessels from Mice
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Brain microvessels isolation from Tg-SwDI mice and C57BL/6 mice was performed using an already established protocol (Lee et al., 2019 (link); Sladojevic et al., 2019 (link)). Briefly, brain tissue was minced in Dulbecco’s Phosphate Buffered Saline and homogenized gently in a Dounce glass homogenizer. Myelin was removed by centrifugation in a 15% Dextran solution (Dextran MW ∼70,000, Sigma Aldrich, St. Louis, MO, United States).
3
Isolation of Peripheral Blood Mononuclear Cells
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This study was approved by the IRB and Ethical Committee, National Taiwan University Hospital, Taipei, Taiwan (201401102RIN). Informed consent was obtained from each participant. Heparinized venous blood was mixed with one-fourth volume of 2% dextran solution (mol. wt. 464,000 daltons) (Sigma-Aldrich Chemical Company, St. Louis, MO, USA) and incubated at room temperature for 30 min. Leukocyte-enriched supernatant was collected and layered on a Ficoll-Hypaque density gradient solution (specific gravity 1.077) (Pharmacia Biotech, Uppsala, Sweden) followed by centrifugation at 250× g for 25 min. The MNCs were aspirated from the interface. The cell concentration of MNCs was adjusted to 2 × 106/mL in 10% fetal bovine serum in RPMI-1640. The MNC suspension contained 90% lymphocytes, 5%–8% monocytes, and 2%–5% other cell populations confirmed by Giemsa stain with a viability greater than 95%, as confirmed by trypan blue dye exclusion.
4
Wafer-Scale Micropatterning of Biomolecules
5
Isolation and Culture of Mouse Brain Endothelial Cells
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Cerebral microvessels from infected and non-infected mice were isolated as described (Ross et al., 2019 (link); Howland et al., 2015 (link)). Briefly, brains were minced with a scalpel-blade and homogenized by passing through a 23-gauge needle. The brain homogenate was mixed in an equal volume of 30% dextran solution (MW 70,000; Sigma-Aldrich) and centrifuged at 10,000 g for 15 min at 4° C. After removal of myelin (top layer), the pellet was resuspended in PBS and passed through a 40 µm cell strainer. The cell strainer was then back-flushed with PBS to collect the microvessel fragments. For in vitro culture of MBECs, microvessels from non-infected mice were incubated with 1 mg/ml Collagenase IV (Gibco) and 200 U/ml of DNAse I (Roche) dissolved in HBSS (Ca2+ and Mg2+) for 1.5–2 hr at 37 ° C. Digested microvessels were washed and seeded in 12-well plates pre-coated with 1% gelatin (Gibco) in EBM-2/EGM-2 medium (Lonza) with 4 μg/ml puromycin (Santa Cruz). The purity of MBECs was determined by CD31 expression (70%–80%) and ZO-1 expression as detailed in Ross et al., 2019 (link). MBECs were challenged with freshly egressed T. gondii tachyzoites (RH-LDM, MOI 4) or LPS (100 ng/ml), for six or 12 h, and processed for PCR.
6
Dextran-Coated Magnetic Particle Synthesis
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Dextran-coated particles were prepared similarly to BIONs using the Massart process. In this process, 9.5 mL of Dextran solution (10.0 g L−1, Sigma Aldrich) was added with 83 mL of iron (II/III) solution (FeCl2 × 4H2O (735 mg), Sigma Aldrich; FeCl3 × 6H2O (2000 mg), Carl Roth) in a 100 mL round bottom flask under N2 atmosphere. Co-precipitation is started after adding 7.5 mL of 25% NH4OH solution, and the reaction was run under homogeneous stirring at 70 °C for 30 min. After completion of the reaction, the excess salts are removed by washing with degassed ddH2O (4×) until a conductivity lower than 200 μS cm−1 is obtained.
7
Isolation and Characterization of Human PMN
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Polymorphonuclear cells (PMN) were obtained from heparinised blood samples. First, PMN were incubated for 45 min with a 1:2 volume of dextran solution (3%) in NaCl (0.9%; Sigma Aldrich, St. Louis, MO, USA), and the supernatant was centrifuged over Fycoll-Hypaque (GE Healthcare, Uppsala, Sweden) for 25 min at 650× g. The resulting pellet was then incubated with lysis buffer to eliminate remaining erythrocytes (5 min at RT) and centrifuged at 250× g. Finally, pellets containing PMN were washed twice and resuspended with Hank´s balanced salt solution (HBSS; Sigma Aldrich, St. Louis, MO, USA) or Roswell Park Memorial Institute (RPMI) culture medium (Sigma Aldrich, St. Louis, MO, USA), depending on their experimental use. In addition, LUNA-FL (Logos Biosystems Inc., Annandale, VA, USA) was used to determine cell count and viability (acridine orange and propidium iodide double stain). A human macrophage-like (U937) cell line was employed for proliferation and viability studies.
8
Neutrophil Isolation from Mouse Blood
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As described previously12 (link),26 (link),39 (link), blood samples were withdrawn into a heparinized syringe from the abdominal inferior vena cava under lethal anesthesia with isoflurane (Forane®; AbbVie GK). In brief, leukocytes were isolated by dextran sedimentation. Blood samples were gently mixed with 6% dextran solution (Sigma-Aldrich, Deisenhofen, Germany) at a 4:1 volume ratio and held vertically for 15 min. After obtaining the supernatant, neutrophils were separated from mononuclear cells by centrifugation using Pancoll for mouse (PAN Biotech GmbH, Aidenbach, Germany) followed by hypotonic lysis of erythrocytes. The resultant cells contained nearly 90% neutrophils, as assessed by microscopy with Wright-Giemsa stain (Wako Pure Chemical Industries, Ltd. Osaka, Japan).
9
Isolation of Pancreatic Islets from Rats
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Islets were isolated from Lewis rats at the age of 8 to 10 wk. In brief, the abdomen of rat was opened under isoflurane anesthesia. The pancreas was distended by infusing collagenase type XI (10 mg; Cat# C9407; Sigma-Aldrich, St. Louis, MO) solved in Hanks’ balanced salt solution (12 mL; Cat# 05906; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) with 0.1% BSA via the common bile duct. Distended pancreas was then excised and transferred to a 50-mL centrifuge tube followed by 37°C incubation for 17 min for digestion. After washing with Hanks’ balanced salt solution/0.1% BSA thrice, the pancreatic islets were separated by discontinuous density gradient separation with the use of dextran solution (Dextran, Cat# 31390; Sigma-Aldrich). Islets were further purified by hand-picking and washed with CMRL-1066 medium (Cat# 11530-037; Gibco, Life Technologies) containing 10% fetal bovine serum (FBS; Cat# 10437-028; Gibco, Life Technologies) thrice with centrifuging at 1200 r/min for 3 min.
10
RNA Extraction and cDNA Synthesis from Blood
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Total RNA was extracted immediately from 5 mL of EDTA—treated peripheral blood of RA patients and controls. Total leucocytes were isolated using 5% dextran solution (Sigma-Aldrich, St. Louis, MO, USA), and the RNA was obtained using TRIzol reagent (InvitrogenTM life technologies, CA, USA), according to the manufacturer’s instructions. The RNA was analyzed on a NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, USA) by measuring the optical density (OD) at 260 nm for the quantity and the OD260/280 ratio for the quality. Before cDNA synthesis, RNA samples were treated with DNAse I (InvitrogenTM Life Technologies, CA, USA). 1 µg of total RNA was subjected to reverse transcription using oligo(dT) and M-MLV reverse transcriptase, as indicated by the manufacturer (Promega, Madison, WI, USA).
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