PBMCs were isolated from heparinized blood samples of patients with grass pollen allergy by means of Ficoll density gradient centrifugation (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). PBMCs (2 × 105) were cultured in triplicates in 96-well plates (Nunclone; Nalge Nunc International, Roskilde, Denmark) in 200 μL of serum-free Ultra Culture medium (BioWhittaker, Rockland, Me) supplemented with l -glutamine (10 mL of 200 mmol/L l -glutamine per liter; Sigma, St Louis, Mo), β-mercaptoethanol (5 mL of 50 mmol/L β-mercaptoethanol per liter, Sigma) and gentamicin (2 mL of 50 mg/mL per liter of medium, Sigma) at 37°C and 5% CO2 in a humidified atmosphere. PBMCs were stimulated with a mix of rPhl p 1, 2, 5, and 6 (0.5 μg per well of each allergen, recombinant mix) or rBet v 1 (2 μg per well) as a control in the presence of heat-inactivated (56°C for 30 minutes) 1:10 diluted sera obtained from rabbits 1 month after the last immunization with BM32 (40-μg dose), Alutard, or Allergovit or from normal rabbits. For control purposes, 4 U of IL-2 per well (Boehringer, Mannheim, Germany) or medium alone was used.
Ficoll density gradient centrifugation
Manufactured by Cytiva
Sourced in Sweden, United Kingdom, Germany
Ficoll density gradient centrifugation is a laboratory technique used to separate cells or other particles based on their density. It involves the use of a Ficoll reagent, which creates a density gradient during centrifugation, allowing different cell types or particles to be isolated based on their specific densities.
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Lab products found in correlation
Ultraculture medium, by Lonza (4 mentions)
L glutamine, by Merck Group (2 mentions)
Nunclone, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Boehringer Ingelheim (2 mentions)
Gentamicin, by Merck Group (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
24 well cell culture plate, by Corning (2 mentions)
Magnetic cell sorting monocyte isolation kit 2, by Miltenyi Biotec (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Roche (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
3h thymidine, by Cytiva (2 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Vle rpmi 1640 medium, by Harvard Bioscience (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Vle rpmi 1640, by Harvard Bioscience (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Foxp3 pe, by BioLegend (1 mentions)
13 protocols using ficoll density gradient centrifugation
1
Allergen-Specific PBMC Stimulation Assay
2
Isolating RNA from Blood Leukocytes
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Ficoll density gradient centrifugation (Amersham Pharmacia Biotech, Tokyo, Japan) was used to separate leukocytes from the blood samples of HCC patients before surgery and of HCs. Then, Qiagen miRNeasy® Mini Kit (Qiagen, Hilden, Germany) was used to extract the total RNA from blood leukocytes and tissues. Ribonucleic acid was kept at -80°C before analysis.
3
Isolation and Cultivation of Human Monocytes
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By using Ficoll density gradient centrifugation (Amersham, Freiburg, Germany), human cord blood mononuclear cells (CBMC) as well as human peripheral blood mononuclear cells (PBMC) were isolated. Afterwards, the cells were washed with PBS, and monocytes were separated from remaining cell types by using the magnetic cell sorting monocyte isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's recommendation. Detected by flow cytometry, the method routinely yielded 95% purity of the population while 90% CD14-positive cells were defined as minimal cut-off value. The cells were standardly cultivated in VLE RPMI-1640 medium (Biochrom, Berlin, Germany) containing 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Germany) and 1% penicillin/streptomycin (Thermo Fisher, Massachusetts, USA). Postphagocytic reaction experiments were performed in 24-well cell culture plates (Costar, Bodenheim, Germany) containing 1 × 106 monocytes/ml.
4
PBMC Proliferation Assay for CM Allergy
5
Isolation and Culture of Human Monocytes
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Isolation of peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) was performed by using Ficoll density gradient centrifugation (Amersham, Freiburg, Germany). Cells were washed and resuspended in VLE-RPMI 1640 (Biochrom, Berlin, Germany). To separate the monocytes, magnetic cell sorting monocyte isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) was used according to the manufacturer's protocol. The minimal purity of the resulting population was defined to be 90% CD14-positive cells, as detected by flow cytometry. For analysis of postphagocytic reactions, 106 cells/ml were seeded in 24-well cell culture plates (Costar, Bodenheim, Germany) in VLE-RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Germany) and 1% penicillin/streptomycin (PenStrep, Thermo Fischer, Massachusetts, USA).
6
Analyzing Human Regulatory T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from 20 to 25 ml of peripheral blood samples via Ficoll density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). According to the recommendation of the international working group of the experts in flow cytometry [7 (link)], CD3, CD4, CD25, CD127, and FoxP3 markers were used to analyze human Treg cells using a gating strategy illustrated in Supplementary Figure 1. Specifically, CD3-FITC, CD45-PerCP, CD127-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD4-Alexa Fluor® 700, (EXBIO a.s., Prague, Czech Republic), Foxp3-PE, and CD25-Brilliant Violet (BioLegend, San Diego, USA) antibodies (Abs) were used. For intracellular staining, we used Foxp3-Fix/Perm and Foxp3-PErm Buffers (BioLegend, San Diego, USA). The flow cytometric data were collected on a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using Summit 4.3 software (Dako, Glostrup, Denmark).
7
PBMC Isolation from Peripheral Blood
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Ten milliliters of peripheral blood containing ethylenediaminetetraacetic acid (EDTA) was collected from both patients and healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were isolated from each peripheral blood sample using Ficoll density-gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). After centrifugation, the PBMCs were collected and washed three times in PBS. The percentage of cell viability was determined using the Trypan blue dye exclusion method, and cells were stored at −80 °C for further analysis.
8
Allergen-Specific PBMC Stimulation Assay
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PBMCs were isolated from heparinized blood samples of patients with grass pollen allergy by means of Ficoll density gradient centrifugation (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). PBMCs (2 × 105) were cultured in triplicates in 96-well plates (Nunclone; Nalge Nunc International, Roskilde, Denmark) in 200 μL of serum-free Ultra Culture medium (BioWhittaker, Rockland, Me) supplemented with l -glutamine (10 mL of 200 mmol/L l -glutamine per liter; Sigma, St Louis, Mo), β-mercaptoethanol (5 mL of 50 mmol/L β-mercaptoethanol per liter, Sigma) and gentamicin (2 mL of 50 mg/mL per liter of medium, Sigma) at 37°C and 5% CO2 in a humidified atmosphere. PBMCs were stimulated with a mix of rPhl p 1, 2, 5, and 6 (0.5 μg per well of each allergen, recombinant mix) or rBet v 1 (2 μg per well) as a control in the presence of heat-inactivated (56°C for 30 minutes) 1:10 diluted sera obtained from rabbits 1 month after the last immunization with BM32 (40-μg dose), Alutard, or Allergovit or from normal rabbits. For control purposes, 4 U of IL-2 per well (Boehringer, Mannheim, Germany) or medium alone was used.
9
Isolation and Characterization of Immune Cell Subsets
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Peripheral blood mononuclear cells [PBMC] were isolated by Ficoll density gradient centrifugation [Amersham Pharmacia Biotech, Uppsala, Sweden]. Lamina propria mononuclear cells [LPMCs] were isolated as previously described.25 (link),26 (link) Mesenteric lymph nodes [LNs] were smashed into 70-μm nylon strainers [BD Biosciences] and erythrocytes lysed with red blood cell [RBC] lysis buffer [BD Biosciences].
For characterisation of T helper [Th] cell subsets from PBMCs, LNs, and LPMCs, T cells were stained with a combination of fluorochrome-conjugated antibodies as specified inSupplementary Table 2 and sorted [FACSAria III, BD Biosciences], according to the expression of the following specific surface markers combination: CD4+IL-7R+CD25lowCCR6-CXCR3+ [Th1 cells], CD4+IL-7R+CD25lowCCR6+CXCR3+ [Th1/17 cells] and CD4+IL-7R+CD25lowCCR6+CXCR3- [Th17 cells]. These three subsets were further subdivided according to CCR5 expression.
Human monocytes for antigen-specificity assays and for generation of monocyte-derived dendritic cells [MoDC] were purified from blood samples by Ficoll density gradient separation and by positive selection using CD14+ selection [CD14 Microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany].
For characterisation of T helper [Th] cell subsets from PBMCs, LNs, and LPMCs, T cells were stained with a combination of fluorochrome-conjugated antibodies as specified in
Human monocytes for antigen-specificity assays and for generation of monocyte-derived dendritic cells [MoDC] were purified from blood samples by Ficoll density gradient separation and by positive selection using CD14+ selection [CD14 Microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany].
10
PBMC Proliferation Assay for CM Allergy
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PBMCs from six nonallergic individuals and seven CM‐allergic patients were isolated from heparinized blood samples by Ficoll density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). PBMCs (2 × 105 cells per well) were cultured in triplicates in 96‐well plates (Nunclone; Nalgen Nunc International, Roskilde, Denmark) in 200 μl serum‐free Ultra Culture medium (BioWhittaker, Rockland, ME, USA) supplemented with 2 mM l ‐glutamine (Gibco, Carlsbad, CA, USA), 50 μM β‐mercaptoethanol (Gibco), and 0.1 mg gentamicin per 500 ml (Gibco). The cells were incubated at 37°C in a humidified atmosphere with 5% CO2 for 7 days and stimulated with different concentrations of milk samples (0.05, 0.5, 3, and 10 μg/well), 4 U IL‐2 per well (Roche) as a positive control and medium alone as a negative control in duplicate. After 6 days of incubation, 0.5 mCi 3H‐thymidine (Amersham, Buckinghamshire, UK) was added to each well for 16 h, and then, the incorporated radioactivity was measured by liquid scintillation counting. Proliferation was expressed as counts per minute (c.p.m.; means of triplicates) using a microbeta scintillation counter (Wallac ADL, Freiburg, Germany). The mean stimulation indices (SI) were calculated as quotient of triplicate c.p.m. with antigen vs medium and shown are the SI obtained by stimulation with 10 μg protein/well.
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