Blood samples were collected from the jugular vein in vacutainer plasma tubes coated with sodium heparin (BD Biosciences, San Jose, CA) according to the recommendations of the United States Department of Agriculture regulations, the National Research Council’s Guide for the Care and Use of Laboratory Animals, as well as all relevant state and federal regulations and policies. Isolation of peripheral blood mononuclear cells (PBMCs) was performed using Ficoll-Paque TM PREMIUM (GE Healthcare Bio-Sciences Corp., Uppsala, Sweden) as previously described (Artiaga et al. 2014 (link)). To obtain total blood leukocytes, 200 μl of whole blood was incubated with an ammonium chloride based buffer to lyse red blood cells.
Ficoll paquetm premium
Manufactured by GE Healthcare
Sourced in Sweden, United Kingdom
Ficoll-PaqueTM PREMIUM is a density gradient medium used for the isolation and purification of cells and biomolecules. It is a sterile, pyrogen-free, and endotoxin-tested solution that facilitates the separation of different cell types based on their density differences.
Automatically generated - may contain errors
Lab products found in correlation
Sodium heparin, by BD (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Human recombinant tgfβ, by R&D Systems (1 mentions)
Il 10, by ProSpec (1 mentions)
Complete rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Sepmatetm tube, by STEMCELL (1 mentions)
Vacutainer, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Collagenase type 3, by Worthington (1 mentions)
Dnase 1, by Zymo Research (1 mentions)
Cd34 microbead kit, by Miltenyi Biotec (1 mentions)
11 protocols using ficoll paquetm premium
1
Isolation of Blood Leukocytes
2
Quantifying Leukocyte Subpopulations in Peripheral Blood
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Peripheral blood (PB) samples from patients were collected on day 1 and day 14 for the identification and quantification of leukocyte subpopulations (Figure 1 ). All patients provided written informed consent to participate in the study. For Innate Lymphoid cells (ILCs) analysis, peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-PaqueTM PREMIUM (GE Healthcare, Chicago, Illinois, United States). A total of 1 x 107 cells were cryopreserved in liquid nitrogen in freezing media (RPMI-1640 50%, FBS 40%, and 10% DMSO) until use for flow cytometry characterization. Serum from patients was collected and conserved at−80°C for cytokine analyses (Figure 1 ).
3
Expansion of Human Hematopoietic Stem Cells
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CB mononuclear cells were prepared by density centrifugation using Ficoll-PaqueTM premium (GE Healthcare) and HSC were isolated using the CD34 MicroBead kit48 (link) and frozen for future use. The purity of CD34+ cells ranged from 90 to 98%. Thawed CD34+ cells were plated on irradiated EL08.1D2 cells and cultured as described by Grzywacz et al.30 (link) except for the addition of 50 ng/mL IL-15 for the last two weeks31 (link). Where indicated, different concentrations of human recombinant TGF-β (R&D Systems) and IL-10 (Prospec) were added.
4
PBMC Isolation and Stimulation Protocol
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Whole blood samples were collected from healthy participants. Human PBMCs were isolated with Ficoll-PaqueTM PREMIUM (17-5442-02, GE Healthcare) from freshly collected peripheral venous blood from healthy donors. Briefly, Ficoll-Paque medium was added to the centrifuge tube, and the diluted blood sample was layered onto the Ficoll-Paque medium solution. The tube was centrifuged at 400 × g for 30 to 40 min at 18 °C to 20 °C with the brake turned off. The upper layer containing plasma and platelets was aspirated using a sterile pipette, leaving the layer of mononuclear cells undisturbed at the interface. The layer of mononuclear cells was washed twice with 1× PBS. For the siRNA knockdown assays, human PBMCs were transfected with RfectSP siRNA Transfection Reagent (BioGenerator Biotechnology) according to the manufacturer’s instructions. Five days after transfection, human PBMCs were stimulated with Curdlan (100 μg/mL), HKCA (MOI = 2) or α-mannan (100 μg/mL) and harvested for gene expression analysis. This study followed the guidelines established by the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. All study participants signed a written informed consent form.
5
Isolation of Peripheral Blood Mononuclear Cells
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PBMCs were separated by Ficoll-Hypaque (Ficoll-PaqueTM PREMIUM, GE Healthcare) density gradient centrifugation either from venous blood collected from healthy young adult volunteers after consent, or from buffy coat residua after platelet separation of blood from anonymized healthy adult blood bank donors (age range 22–45 years) of All India Institute of Medical Sciences, New Delhi.
6
Umbilical Blood MNC Isolation Protocol
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Donors undergoing delivery were recruited in the study. Samples were harvested from normal term pregnancies (n = 26) between 37 and 42 weeks of gestation, both after vaginal or cesarean section delivery. The umbilical blood was allowed to flow into heparinized tubes (5,000 IU/mL) and processed within 12 h. Samples were then diluted with D-PBS (Thermo Fisher Scientific) and MNCs collected by density gradient centrifugation using Ficoll-PaqueTM PREMIUM (GE Healthcare) and cultured as described above applying the protocol validated for BM-MNCs and the same PhABS batch described above.
7
Isolation and Stimulation of Human PBMC
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Venous blood was collected using BD Vacutainer® (367874) containing sodium heparin as an anticoagulant, in accordance with a protocol approved by the University of Manitoba Ethics Review Board. Blood was diluted 1:1 with complete RPMI1640 medium (Gibco, Invitrogen, Canada), containing 1 mM sodium pyruvate and 10% (v/v) FBS. Human PBMC was separated over Ficoll-PaqueTM Premium (GE Healthcare, Buckinghamshire, UK) using SepMateTM tubes (Stemcell Technologies, Vancouver, BC) according to the manufacturer’s instructions. Briefly, Ficoll-PaqueTM Premium was pre-layered into the bottom of the SepMateTM tubes through the insert. Diluted blood was carefully pipetted into the SepMateTM tube and centrifuge for 1000 × g for 10 min at room temperature (RT). The top layer containing the enriched PBMC was collected into a new centrifuge tube and washed twice in complete RPMI 1640 medium (300 × g for 10 min). PBMC (1 × 106 cells/mL) were cultured in 6- or 24- well tissue culture plates, rested at 37 °C in a humidified 5% CO2 incubator for 1 hour before stimulations with toll-like receptor ligands; MTB lipoprotein (100 ng/ml), poly I:C (1 μg/ml), bacterial lipopolysaccharide (100 ng/ml), flagellin (100 ng/ml), and R848 (1 μg/ml), for 6 h or 24 h as indicated.
8
Isolation and Sorting of Naive CD4 T Cells
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Following euthanasia, single cell suspensions of mouse spleen cells were prepared, subjected to red cell lysis, and stained with anti-mouse CD4, CD25, CD44 and CD62L Abs and electronically sorted (FACSAria III, BD) for naïve CD4 T cells (CD4 + CD25-CD44-CD62L+). For sorting NCD4lo and NCD4hi cells, further gating was done for the approximately 10% NCD4lo and approximately 10% NCD4hi populations in the NCD4 gated population. For sorting CD4lo and CD4hi mature single positive thymic cells, thymic single cell suspensions were made from young B6 mice, stained with CD4, CD8, CD24 and Qa2. CD4 + CD8-CD24-Qa2+ cells identified as mature CD4 cells were further gated for CD4lo and CD4hi and sorted as for peripheral cells.
Blood from healthy, young, consenting adults between the ages of 22 and 35 years (n = 10) with equal representation of men and women was collected and PBMCs separated from heparinized blood by density gradient centrifugation using Ficoll-PaqueTM PREMIUM (GE Healthcare Biosciences AB, Little Chalfont, UK). For sorting human NCD4hi and NCD4lo cells, CD4 + CD25-CD45RA+ low forward scatter cells were gated for NCD4lo and NCD4hi as described for mouse cells and sorted. After sorting, individual fractions were >98% pure in all cases.
Blood from healthy, young, consenting adults between the ages of 22 and 35 years (n = 10) with equal representation of men and women was collected and PBMCs separated from heparinized blood by density gradient centrifugation using Ficoll-PaqueTM PREMIUM (GE Healthcare Biosciences AB, Little Chalfont, UK). For sorting human NCD4hi and NCD4lo cells, CD4 + CD25-CD45RA+ low forward scatter cells were gated for NCD4lo and NCD4hi as described for mouse cells and sorted. After sorting, individual fractions were >98% pure in all cases.
9
Isolation and Culture of Bone Marrow-Derived Mesenchymal Stem Cells
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BM MSCs were isolated as previously described [18 (link)]. Extracted bone marrow samples (n = 9) from 5 males and 4 females were diluted twice with calcium- and magnesium-free phosphate-buffered saline (DPBS) and layered on top of Ficoll-PaqueTM Premium (GE Healthcare, Uppsala, Sweden) at a ratio of 1:2. This was then centrifuged at 400 g (with brake off) for 30 min at 20 °C. The uppermost layer was aspirated and discarded. The mononuclear layer was collected and diluted three times with DPBS. This was centrifuged at 400 g for 5 min and washed again with DPBS. The supernatant was discarded, and the pellet was resuspended with 10 mL of growth medium (low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/ml streptomycin). This was then centrifuged at 400 g for 5 min. The cellular pellet was resuspended in a growth medium. The cells were seeded onto conventional tissue culture plates at a concentration of 1 × 106 cells/cm2 and incubated in a 5% CO2 incubator with humidified air at 37 °C. The medium was replaced every 3 days. When cells reached 80% confluence, they were split at a ratio of 1:4. BM MSCs at passage 3 (P3) were used for all experiments.
10
Isolating Immune Cells from Biological Samples
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Blood samples were collected from the jugular vein in vacutainer plasma tubes coated with sodium heparin (BD Biosciences, San Jose, CA). Isolation of peripheral blood mononuclear cells (PBMCs) was performed using standard procedures with Ficoll-PaqueTM PREMIUM (GE Healthcare BioSciences Corp., Uppsala, Sweden) as previously described25 (link). Spleen and lymph node tissues were collected and dispersed into single cell suspensions using closed sterile tissue grinders as previously described25 (link). Lung tissues were minced using scissors, digested with a mixture of 2 U/ml DNase I (Zymo Research, Irvine, CA) and 250 U/ml collagenase type III (Worthington Biochemical, Worthington, NJ) at 37 °C for 45 minutes and filtered through a 100 μm cell strainer. BALF was filtered using a 100 μm cell strainer. An ammonium chloride-based lysis buffer was used to remove residual red blood cells from blood and tissue samples.
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