Blood samples were taken just before and within a month after each CT examination (Table 1 ). Mononuclear blood cells were isolated from heparinized PB from each sample using BD Vacutainer CPT tubes (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Cells were suspended in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) containing 20% fetal bovine serum (Equitech Bio, Keilor East, Australia), 2% phytohaemagglutinin-HA15 (Remel, Lenexa, KS, USA) and 60 μg/ml kanamycin solution (Life Technologies, Carlsbad, CA, USA) in a tube. Lymphocytes were cultured in a 5% humidified CO2 incubator at 37°C for 46 h. Then, colcemid solution (Wako, Osaka, Japan) was added (final concentration: 50 ng/ml or 0.05 μg/ml) and cells were cultured for an additional 2 h. After 48 h of culture, chromosome preparations were made according to the standard cytogenetic procedure [11] .
Vacutainer cpt tube
Manufactured by BD
Sourced in United States, Germany
The BD Vacutainer CPT tubes are a type of blood collection tube used for the separation and isolation of peripheral blood mononuclear cells (PBMCs) from whole blood. These tubes contain a polyester gel and a density gradient medium to facilitate the separation of blood components during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Rpmi 1640 medium, by Nacalai Tesque (5 mentions)
Kanamycin solution, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Equitech-Bio (5 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Ficoll hypaque density gradient centrifugation, by GE Healthcare (4 mentions)
Ammonium chloride buffer, by Lonza (4 mentions)
Ammoniumchloride lysis buffer, by Lonza (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Colcemid, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Rock inhibitor y 27632, by Selleck Chemicals (3 mentions)
Cytotune ips 2.0 sendai reprogramming kit, by Thermo Fisher Scientific (3 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (3 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (3 mentions)
Stempro 34 sfm medium, by Thermo Fisher Scientific (3 mentions)
175 protocols using vacutainer cpt tube
1
Lymphocyte Isolation and Metaphase Preparation
2
Gamma-Ray Irradiation Protocol for Lymphocyte Analysis
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PB samples were irradiated with gamma-rays (Gamma cell 40, Best Theratronics, Ottawa, Ontario, Canada; installation date: March, 2009.) at eight doses (0, 10, 20, 50, 100, 200, 500 or 1000 mGy). Plastic microtubes containing whole blood were irradiated at a distance of 16 cm at room temperature with gamma-rays from a 60Co radiation source (1.11 TBq) at a dose rate of 26.26t (time: min) + 6.42 mGy per min, where 26.26 is the dose rate and 6.42 is the dose to the sample entering and leaving the irradiation source. The doses were measured using an ionization chamber detector for gamma-rays.
Mononuclear blood cells were isolated from heparinized PB samples using BD Vacutainer CPT tubes (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Cells were suspended in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) containing 20% fetal bovine serum (Equitech Bio, Keilor East, Australia), 2% phytohaemagglutinin-HA15 (Remel, Lenexa, KS, USA) and 60 μg/ml of kanamycin solution (Life Technologies, Carlsbad, CA, USA) in a 6-well plate. Lymphocytes were cultured in a 5% humidified CO2 incubator at 37°C for 48 h. Colcemid solution (Wako, Osaka, Japan) was added (final concentration: 0.015–0.02 μg/ml) 2 h before cell harvest, then chromosome preparations were made according to a standard cytogenetic procedure [23 ].
Mononuclear blood cells were isolated from heparinized PB samples using BD Vacutainer CPT tubes (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Cells were suspended in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) containing 20% fetal bovine serum (Equitech Bio, Keilor East, Australia), 2% phytohaemagglutinin-HA15 (Remel, Lenexa, KS, USA) and 60 μg/ml of kanamycin solution (Life Technologies, Carlsbad, CA, USA) in a 6-well plate. Lymphocytes were cultured in a 5% humidified CO2 incubator at 37°C for 48 h. Colcemid solution (Wako, Osaka, Japan) was added (final concentration: 0.015–0.02 μg/ml) 2 h before cell harvest, then chromosome preparations were made according to a standard cytogenetic procedure [23 ].
3
Isolation of Healthy Donor PBMCs
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Blood samples of healthy donors were collected in cell preparation tubes with sodium citrate (BD Vacutainer CPT Tubes, BD Biosciences, Franklin Lakes, NJ, USA). PBMCs were obtained by centrifugation following the manufacturer’s protocol.
4
Characterizing Immune Cells in Blood
5
Isolation of PBMCs from Vitiligo Patients
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Blood was collected from 4 volunteers and 4 vitiligo patients between 8:00 and 12:00 in the morning to limit the effect of circadian variation of cytokine production. BD Vacutainer CPT tubes (BD, New York, N.Y., USA) were used to separate PBMCs from other blood cells. The cells were centrifuged at 1,500 g for 30 min at 20°C. After that, blood sera were collected from the top of the PBMCs. Phosphate-buffered saline was used to wash the isolated PBMCs twice, after which they were centrifuged at 190 g for 10 min at 20°C. The supernatant was collected and the cells were stored at −80°C until RNA extraction.
Total RNA was extracted applying the mirVana miRNA Isolation Kit (Ambion, Life Technologies Corp., Carlsbad, Calif., USA) according to the manufacturer's protocol. RNA quality was assessed using Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit (Agilent Technologies Inc., Calif., USA). The RNA integrity number was over 9 for all samples.
Total RNA was extracted applying the mirVana miRNA Isolation Kit (Ambion, Life Technologies Corp., Carlsbad, Calif., USA) according to the manufacturer's protocol. RNA quality was assessed using Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit (Agilent Technologies Inc., Calif., USA). The RNA integrity number was over 9 for all samples.
6
PBMC Isolation and Preservation
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PBMCs were isolated using BD Vacutainer CPT tubes, washed, and resuspended in RPMI 1640 with 10% FCS (v/v) for immediate use or frozen in fetal calf serum with 10% dimethyl sulfoxide (v/v) for subsequent analysis. Plasma samples were saved at −80°C for subsequent analysis.
7
Plasma Lipid Extraction for LC-MS Analysis
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Patient blood was collected by regular phlebotomy directly into BD Vacutainer® CPT™ tubes (heparin-Ficoll tubes). Plasma was separated by centrifugation at 3000 rpm for 20 min. Lipid extraction was performed for liquid chromatography–mass spectrometry (LC–MS) analysis as we previously described with slight modifications39 (link). Briefly, 20 µL ice-cold methanol contained internal standards and butylated hydroxytoluene (BHT) was added into 50 µL of each plasma sample. After vortex for 5 s, sample mixtures were placed on ice for 5 min before vortex again for another 30 s. After that, samples were incubated for 5 min at 1500 rpm at 4 °C in an orbital mixer before centrifugation at 4500 rpm for 10 min at 4 °C. The lipid-containing bottom phase was transferred to glass vials and lyophilized using Centrivap cold trap (Labconco) and stored at −80 °C before analysis.
8
Techniques for Isolating White Blood Cells
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Six techniques to separate white blood cells from erythrocytes were evaluated, including erythrocyte lysis buffer, ammonium chloride lysis buffer, distilled water, BD Vacutainer® CPT™ tubes, dextran, and lymphocyte separation medium, as described below. Following erythrocyte lysis, white blood cell pellets were suspended in various diluents (1× TE buffer, 1× PBS, 0·05× PBS, animal product-free LB Lennox medium (APF-LB; Athena Environmental Sciences, Baltimore, MD) and 1× TE buffer plus 5 g l−1 of NaCl), as necessary.
9
Blood Cell Separation and Analysis
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All blood samples were collected in BD Vacutainer® CPT™ tubes (BD Bioscience, San Jose, CA). Mononuclear white blood cells (WBCs), plasma, and red blood cells (RBCs) were separated from the CPT tubes within an 8 h window, according to the manufacturer’s instructions. Hemoglobin status was determined using cellulose acetate membrane electrophoresis at the Department of Hematology at the Korle-Bu Teaching Hospital (21 (link)). Hematological characteristics were assessed by CBC at the hospital’s clinical pathology and hematology laboratories. Malaria status was determined using Plasmodium falciparum Rapid Diagnostic Test kits and thick smear microscopy. HIV status was also assessed using HIV Rapid Diagnostic Tests.
10
PBMC Isolation and Cryopreservation
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Blood was obtained in 8-ml BD Vacutainer™ CPT ™ tubes with sodium heparin, and peripheral blood mononuclear cells (PBMCs) were isolated according to the manufacturer's instructions. Freezing medium [10% dimethylsulphoxide (DMSO) and 90% fetal calf serum (FCS)] was added, and the cells were stored at −135°C. Plasma was collected and frozen at −80°C.
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