Pig blood was collected on 1.3 M citrate and mononuclear cells were isolated on Ficoll-Hypaque density gradient (Amersham Bioscience) and used fresh. Spleen cells were isolated by mechanical dissociation followed by filtration on successive 500 µm and 100 µm mesh-sized nylon filters and centrifugation on Ficoll-Hypaque density gradient. Spleen cells were step frozen in FCS + 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen. Migrated cells from pig skin explants were obtained from 24 punch biopsies (8 mm), which were floated for 24 h on 10 ml RPMI 1640 medium + 10% FCS and antibiotics. Migrated cells were collected after loading on a 100 µm mesh-sized nylon filter and used fresh. Alveolar macrophages were collected as described, step frozen in FCS + 10% DMSO, and stored in liquid nitrogen (33 (link)). Broncho-alveolar lavage (BAL) was performed twice on isolated left lung with 250 ml PBS + 2 mM EDTA and the collected cells were step frozen and stored in liquid nitrogen.
Ficoll hypaque density gradient
Manufactured by Cytiva
Sourced in Sweden, United States
Ficoll-Hypaque density gradient is a laboratory reagent used for the separation and isolation of cells, such as lymphocytes, from a heterogeneous cell population. It is a sterile, endotoxin-tested solution that creates a density gradient to facilitate the separation of different cell types based on their density.
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Lab products found in correlation
Rpmi 1640, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Diva software, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd19 pe, by Thermo Fisher Scientific (1 mentions)
Cd4 percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd27 apc, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Anti cd3 antibody, by BD (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Easysep human cd4 t cell enrichment kit, by STEMCELL (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Recombinant human interleukin il 1α, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
13 protocols using ficoll hypaque density gradient
1
Isolation of Porcine Immune Cells
2
Profiling Immune Cell Phenotypes and Metabolic Parameters in Aging Adults
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Blood was collected from adults (n=6, four females and 2 males) aged 64-67 years in 2015. Peripheral blood mononuclear cells were isolated using a Ficoll–Hypaque density gradient (Amersham Biosciences) and centrifugation. Viable cells were counted, adjusted to 2×106/100 μL in 80% fetal bovine serum and 20% dimethylsulfoxide (Sigma), and frozen stored until the phenotyping. In 2021, cells were thawed, checked for viability, and stained with monoclonal antibodies to the T cell phenotypes CD4 PerCP Cy5.5, CD8 APC Cy7, CD27 APC, CD45RA PE; B cell phenotypes CD19 PE, CD27 APC, IgD PE Cy5.5 (eBioscience), and ACE CD143 fluorescein isothiocyanate (R&D Systems). After 30 minutes of incubation with monoclonal antibodies in the dark at 4 °C, the cells were washed with phosphate-buffered saline and centrifuged. Living cells (based on forward and side scatter) were acquired in the BD FACSCanto II flow cytometry system using the DIVA software (Becton Dickinson).
For assessing metabolic parameters, the serum of studied individuals was previously isolated through centrifugation and frozen stored until use. Measurement of metabolic parameters was performed in the Laboratório Central–Hospital São Paulo, Federal University of São Paulo.
For assessing metabolic parameters, the serum of studied individuals was previously isolated through centrifugation and frozen stored until use. Measurement of metabolic parameters was performed in the Laboratório Central–Hospital São Paulo, Federal University of São Paulo.
3
PBMC Isolation and Cryopreservation
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Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation (Amersham–Pharmacia, Uppsala, Sweden), washed three times with phosphate-buffered saline (GIBCO BRL) and cryopreserved in 90% heat-inactivated foetal calf serum (JRH Biosciences, Kansas, USA) and 10% DMSO (Sigma-Aldrich, Castle Hill, Australia).
4
Measuring Immunosuppressive Effects of MSCs
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To test the inhibitory effects of T1D-MSCs and C-MSCs on allogeneic lymphocyte proliferation, the carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen LifeTechnologies) dilution method was used. Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were separated by Ficoll-Hypaque density gradient (Amersham-Pharmacia), labeled with CFSE (10 μM, for 10 minutes at 37 °C), and resuspended in RPMI 1640 medium (Gibco) supplemented with 5 % human serum albumin (Vialebex® 200 mg/ml; LFB, Rio de Janeiro, Brazil). CFSE-labeled PBMCs were added to the wells containing previously adhered patient or control MSCs, in six different ratios (MSCs:PBMCs = 1:2, 1:5, 1:10, 1:20, 1:50, and 1:100) in the presence of 0.5 μg/ml phytohemagglutinin (PHA; Sigma‐Aldrich, St. Louis, MO, USA). The cocultures were incubated for 5 days at 37 °C with 5 % CO2. Subsequently, PBMCs were harvested, stained with anti‐CD3 antibody (BD, San Jose, CA, USA) and the dilution of CFSE in CD3+ T cells was analyzed by flow cytometry using FACSCalibur™ (BD) equipment.
5
Cryopreservation and Thawing of PBMC
6
Isolation and Sorting of CD4+ T Cells
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Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll‐Hypaque density gradient (Amersham Pharmacia Biotech) from peripheral blood of healthy volunteers, after informed consent with the approval of the Ethics Committee of Lisbon Academic Medical Centre.
CD4 T cells were negatively selected using the EasySep Human CD4+ T‐cell enrichment kit (StemCell Technologies), stained for CD3, CD4, CD25, and CD127, and subsequently sorted into Treg and Tcon cells using a FACSAria flow cytometer (BD Biosciences).
CD4 T cells were negatively selected using the EasySep Human CD4+ T‐cell enrichment kit (StemCell Technologies), stained for CD3, CD4, CD25, and CD127, and subsequently sorted into Treg and Tcon cells using a FACSAria flow cytometer (BD Biosciences).
7
Autologous CIK Cell Preparation Protocol
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Autologous CIK cells were prepared as previously described [14 (link)]. Briefly, peripheral blood mononuclear cells (PBMCs) from participants were obtained from whole blood by centrifugation over Ficoll-Hypaque density gradient (Ficoll-Paque Plus; Amersham Biosciences). The PBMCs (2 × 106 cells/mL) were incubated in fresh RMPI-1640 medium (HyClone, USA) containing 10% serum and 1000 U/mL recombinant human gamma-interferon (IFN-γ, PeproTech, USA). After incubation for 24 h, anti-CD3 antibody (100 ng/mL, BioLegend, USA), recombinant human interleukin- (IL-) 1α (100 U/mL, PeproTech, USA), and recombinant human IL-2 (1000 U/mL, PeproTech, USA) were added to the medium. Cells were incubated and fed every 2 days in fresh complete medium supplemented with rhIL-2 (1000 U/mL) and maintained at a density of 2 × 106 cells/mL.
8
Cell Lines and PBMC Isolation for Cytotoxicity Studies
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Colon cancer cell lines HCT116 (p53+/+), HCT116 (p53−/−) and HT29 (p53mt), breast cancer cell lines MDA-MB-231 (p53mt) and MCF-7 (p53+/+) were obtained from NCCS, India. Blood collected from healthy human volunteers with informed consent (Institutional Review Board 1382) was centrifuged over Ficoll-Hypaque density gradient (Amersham Pharmacia, Uppsala, Sweden) to obtain total peripheral blood mononuclear cells (PBMC)48 (link). Cells were routinely maintained in complete DMEM/RPMI 1640 at 37 °C in a humidified incubator containing 5% CO2. Cells were allowed to reach confluency before use. Dose-dependent and time-dependent cell death experiments were performed using Trypan blue-exclusion test49 (link). All experiments were performed in accordance with approved guidelines and regulations of the Helsinki Declaration (http://www.wma.net/en/30publications/10policies/b3/index.html ). All experimental protocols were approved by informed consent (IRB-1382), Human Ethics Committee, Bose Institute (Approval No: BIHEC/2010-11/2).
9
Isolation and Culture of K562 Cells and Primary CML CD34+ Cells
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The human myelogenous leukaemia cell line K562 were obtained from America Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 (Sigma, St. Louis, MO, USA) medium containing 10% heat-inactivated fetal calf serum (FCS, Hyclone, Logan, UT, USA), 100 unit/ml penicillin and 100 μg/ml streptomycin in a humidified 5% CO2 atmosphere at 37°C.
The bone marrow (BM) samples were obtained from healthy donor or CML patients undergoing diagnostic procedures at Peking university first hospital. Written informed consent was obtained from each healthy donor and CML patient. All the procedures were approved by the Ethics Committee of Beijing Institute of Radiation Medicine. Mononuclear cells were isolated from heparinized samples by centrifugation through a Ficoll-Hypaque density gradient (Amersham Biosciences, Piscataway, NJ, USA). Then, CD34+ cells were isolated by using human CD34 positive selection kit (Stem Cell Technology, Vancouver BC, Canada).
The bone marrow (BM) samples were obtained from healthy donor or CML patients undergoing diagnostic procedures at Peking university first hospital. Written informed consent was obtained from each healthy donor and CML patient. All the procedures were approved by the Ethics Committee of Beijing Institute of Radiation Medicine. Mononuclear cells were isolated from heparinized samples by centrifugation through a Ficoll-Hypaque density gradient (Amersham Biosciences, Piscataway, NJ, USA). Then, CD34+ cells were isolated by using human CD34 positive selection kit (Stem Cell Technology, Vancouver BC, Canada).
10
Characterizing Immune Profiles in HIV Cohorts
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Blood samples were collected at enrollment on tubes with EDTA and centrifuged to separate plasma, which was stored at −80 °C until use. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Amersham, Sweden) and cryopreserved. For those PHI patients that remained ART naïve, additional samples were obtained at a median of 330 days p.i (see Table 1 ). For Chronics, ECs and PHIs, plasma VL (branched-DNA, Versant HIV-1 RNA 3.0 assay; Siemens Healthcare) and CD4/CD8 counts (flow cytometry double platform, BD FACSCanto; BD Biosciences) were determined.
Subsequent functional assays were performed, according to sample availability, using only thawed cells that showed >95% viability after overnight rest in complete RPMI medium [RPMI-1640 (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 2 mM L-glutamine (Sigma-Aldrich, USA), 100 U/ml penicillin (Sigma-Aldrich, USA), 100 mg/ml streptomycin (Sigma-Aldrich, USA), and 10 mM HEPES (Gibco, USA)].
Subsequent functional assays were performed, according to sample availability, using only thawed cells that showed >95% viability after overnight rest in complete RPMI medium [RPMI-1640 (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 2 mM L-glutamine (Sigma-Aldrich, USA), 100 U/ml penicillin (Sigma-Aldrich, USA), 100 mg/ml streptomycin (Sigma-Aldrich, USA), and 10 mM HEPES (Gibco, USA)].
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