PBMCs were isolated from the peripheral blood of 3 HDs using Lymphoprep (Cosmo Bio) according to the manufacturer’s instructions. The cells were stimulated with 1 μM of LF9 peptide or GK12 peptide (negative control) on days 0 and 7, and 50 U/mL rhIL-2 (Peprotech) was added on day 1. TILs or stimulated HD PBMCs were prestained with human FcR blocking reagent (Clear Back, MBL) on day 15 and stained with LF9– or HIVenv584-592–HLA-A24 tetramer (a tetramer for HIV-1 envelope 584-592 aa and HLA-A*24:02, MBL) conjugated with PE or FITC for 20 minutes at 4°C, followed by anti–CD8-PC5 (SFCI21Thy2D3, Beckman Coulter) for 20 minutes at 4°C. The cells were also stained with anti–CD8-APC (HIT8a, BioLegend), anti–CD4-PE-Cy7 (OKT4, BioLegend), and anti–CD3-PE (UCHT1, BioLegend) for 20 minutes at 4°C. The stained cells were analyzed using FACSCanto II with FACSDiva (BD Biosciences). LF9– and HIVenv584-592–HLA-A24 tetramers were purchased from MBL. Synthetic peptides (LF9, GK12) with > 80% purity were purchased (MilliporeSigma and Cosmo Bio, respectively).
Lymphoprep
Manufactured by Cosmo Bio
Sourced in Japan, United States
Lymphoprep is a density gradient medium used for the isolation of mononuclear cells from whole blood or bone marrow. It facilitates the separation of mononuclear cells, including lymphocytes and monocytes, from other blood components through centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Heracell 150i, by Thermo Fisher Scientific (3 mentions)
Np sc1000, by Nipro (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Rhil 2, by Thermo Fisher Scientific (1 mentions)
Anti cd4 pecy7 okt4, by BioLegend (1 mentions)
Anti cd3 pe ucht1, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Easysep human monocyte enrichment kit, by STEMCELL (1 mentions)
Macrophage csf m csf, by BioLegend (1 mentions)
Accutase cell detachment solution, by Innovative Cell Technologies (1 mentions)
Ifn γ, by Sino Biological (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Cell scraper, by Corning (1 mentions)
Ly6g bv605, by BioLegend (1 mentions)
Trustain fcx, by BioLegend (1 mentions)
Cd45 bv711, by BioLegend (1 mentions)
Cd11b pe, by BioLegend (1 mentions)
16 protocols using lymphoprep
1
Characterization of LF9-specific CD8+ T Cells
2
Isolation of Primary AML Blasts
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Primary AML blasts were obtained from 31 newly diagnosed AML patients with different French-American-British (FAB) subtypes, except for FAB M3 (APL). Prior to clinical therapy, mononuclear cell fractions were isolated from freshly collected bone marrow aspirates or peripheral blood using Lymphoprep™ (Cosmo Bio Co., Ltd., Tokyo, Japan), and the cell fractions were then cryopreserved in liquid nitrogen until use. Prior to conducting the experiments, aliquots of the AML cells were rapidly thawed at 37 °C and washed with PBS. Cells were then stained with Wright-Giemsa solution to confirm blastoid cell content and more than 90 % of blastoid cell fraction was ensured in all samples. The study was approved by the Internal Review Committee of Nihon University Itabashi Hospital.
3
Isolation of Mononuclear Cells from Peripheral Blood
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Peripheral blood was diluted 1:1 with saline solution (SS; 0.9% NaCl) and corresponding volume of sterile Lymphoprep™ (Cosmo Bio USA, Inc., Carlsbad, CA, USA) was used. The mixture was centrifuged at 355 × g/room temperature for 30 min. Subsequently, the intermediate layer formed was separated, which corresponds to mononuclear cells, and then washed with an equal amount of SS and centrifuged for 20 min at 355 × g/room temperature. Finally, the supernatant was decanted and the pellet was resuspended in 1 ml of SS ready for counting.
4
Isolation of PBMCs from Microminipigs
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Peripheral blood samples from Microminipigs (S1 Table ) were collected into separate heparinized tubes and centrifuged on Lymphoprep (Axis-Shield, Oslo, Norway) at 670 x g for 30 min. The peripheral blood mononuclear cells (PBMCs) were collected and washed with 30 ml of 1% (w/v) bovine serum albumin (BSA) containing phosphate-buffered saline (PBS). The remaining erythrocytes were lysed osmotically. The PBMCs were washed with PBS and used for further experiments. The single human PBMC sample was purchased from Cosmo Bio Co. Ltd (Tokyo Japan), and the mouse and marmoset PBMCs were collected using Lymphoprep, as described above.
5
Isolation and Differentiation of Primary Immune Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy individuals and patients by density gradient centrifugation with Lymphoprep (#AXS-1114547; Cosmo Bio USA, CA, United States). Monocytes isolated from PBMCs were isolated with EasySep Human Monocyte Enrichment Kit (#19058; Stemcell Technologies), or by plastic adherence as previously described.19 (link) Monocytes were differentiated into Mφ by treatment with 20 ng/mL macrophage-CSF (M-CSF) (#574806; BioLegend, CA, United States) for 5-day as reported.19 (link) Mφ were further differentiated by stimulating with 100 U/mL IFN-γ (#11725-HNAS; Sino Biological; PA, United States) and 100 ng/mL LPS (#L2630; MilliporeSigma, MA, United States). Mφ were detached from plates using Accutase Cell Detachment Solution (#AT-104; Innovative Cell Technologies, CA, United States) and Cell Scraper (#3010; Corning, NY, United States). Naive CD4 T cells and total CD4 T cells were purified using the EasySep human naive CD4 T cell and human CD4 T cell isolation kits (#19555, #17952; Stemcell Technologies, Vancouver, Canada). Cells were cultured in RPMI 1640 medium (#11875135; Thermo Fisher Scientific, MA, United States) supplemented with 10% FBS (#100–106; GeminiBio, CA, United States) and were incubated at 37°C with 5% CO2.
6
LPS-Induced Neutrophil Responses in Mice
7
PBMC Isolation from Blood Samples
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Blood samples obtained from a healthy donor and mCRC patients were collected in tubes containing anticoagulant solution, heparin and citrate phosphate dextrose (NP-SC1000, Cat#31–620, Nipro, Osaka, Japan), respectively. The PBMCs were then isolated by centrifugation on Lymphoprep™ (Cat#1114544, CosmoBio, Tokyo, Japan).
8
Isolation of T cells, B cells, and Monocytes
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Mononuclear cells were harvested from the peripheral blood of healthy volunteers, and neutrophils and mononuclear cells were separated with sodium diatrizoate and polysaccharide (Lymphoprep; COSMO BIO, Inc., Tokyo, Japan) according to the manufacturer's instructions. Then, the mononuclear cells were separated into T cells (Pan T Cell Isolation Kit, human; Miltenyi Biotec, Bergisch Gladbach, Germany), B cells (Pan B Cell Isolation Kit, human; Miltenyi Biotec), and monocytes/macrophages (CD14 MicroBeads, human; Miltenyi Biotec) by magnetic cell sorting (MACS cell separation; Miltenyi Biotec) according to the manufacturer's protocol.
9
Isolation of Mononuclear Cells from Blood
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Blood samples were transferred to 5-ml tubes containing anticoagulant with EDTA, and were diluted by the addition of an equal volume (3 ml) of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS). Next, 6 ml of the diluted blood sample was subsequently overlaid on a 4-ml Lymphoprep™ (Cosmo Bio Co, Tokyo, Japan) placed in a 15-ml centrifuge tube. The mixing of blood and separation fluid was avoided, and the tube was capped to prevent the formation of aerosols. The tubes were spun at 800 × g for 20 min at room temperature in a swing-out rotor centrifuge. After spinning, mononuclear cells were removed from the distinct band at the sample/medium interface using a Pasteur pipette without disturbing the upper layer. Mononuclear cells were diluted in 2 ml PBS containing 2% FBS, and the cells were subsequently pelleted by spinning at 250 × g for 5 min at 25°C.
10
Analyzing TKI-Induced Blood Changes
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Peripheral blood samples were collected before and after TKI intake. Sample collection after TKI dosage was performed 1 h after dasatinib treatment or 2 h after imatinib or nilotinib treatment, which is considered the peak concentration time as described previously 12 . Complete blood cell counts and the percentage of each leukocyte subset were evaluated in all samples. For analysis of signal transduction pathways using the phospho‐flow method, mononuclear cell fractions were isolated from freshly collected peripheral blood using Lymphoprep™ (Cosmo Bio Co., Ltd., Tokyo, Japan) to classify each cellular subset precisely by reducing the effect of outliers.
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