PBMCs were purified from heparinized peripheral whole blood using a Ficoll–Histopaque gradient (1.077 g/ml; GE Healthcare Life Sciences, Piscataway, NJ, USA). They were stored in liquid nitrogen in a freezing medium consisting of 50% FBS, 10% dimethyl sulfoxide (DMSO), and 40% RPMI-1640 (all from Thermo Fisher Scientific) until analysis (27 (link)). Cells were cultured in complete RPMI-1640 containing 10% FBS and 1× penicillin/streptomycin (Thermo Fisher Scientific) and stimulated as follows.
Ficoll histopaque gradient
Manufactured by GE Healthcare
Sourced in United States
Ficoll Histopaque gradient is a laboratory product used for the separation and purification of cells, primarily mononuclear cells, from whole blood or other biological samples. It is a density gradient medium that allows for the efficient isolation of specific cell populations through centrifugation.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Heparin, by BD (2 mentions)
Percoll gradient, by GE Healthcare (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Untouched human t cell isolation kit, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
L glutamine, by Corning (2 mentions)
Calcium chloride, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
70 m cell filter, by BD (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Il 17, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by American Type Culture Collection (1 mentions)
Anti il 4, by BioLegend (1 mentions)
Il 17, by R&D Systems (1 mentions)
Soluble anti cd28, by BD (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
8 protocols using ficoll histopaque gradient
1
Cryopreservation and Stimulation of PBMCs
2
Isolation of Mononuclear Cells from Primate Samples
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PBMCs were isolated from whole blood anticoagulated with heparin (132 Units per 8 ml blood) (BD Biosciences, Oxford, UK) using standard methods. Of note is that the material used for density gradient centrifugation was adjusted dependent on the macaque species, with a Ficoll Histopaque gradient (GE Healthcare, USA) used with rhesus macaque blood and a Percoll gradient (GE Healthcare) used with cynomolgus macaques. Mononuclear cells (MNC) were isolated from spleen and lung tissue samples using an OctoMACS tissue dissociation device (Miltenyi Biotec). Lung tissue samples were dissected into approximately 5mm3 pieces and incubated for 1 h in a solution of 772.8 U/ml collagenase + 426 U/ml DNase (both from Sigma) diluted in Earle’s balanced salt solution supplemented with 200 mg/ml Calcium Chloride (Gibco, Life Technologies, Renfrew, UK), at 37 °C with continual gentle mixing of the tube. The homogenised solution was passed through a 70 µm cell filter (BD Biosciences) and the mononuclear cells separated by Ficoll Histopaque density gradient centrifugation. PBMCs and MNC isolated from tissues were stored at −180 °C until resuscitated for analysis.
3
Isolation and Activation of CD3+ T Cells
4
Isolation and culture of immune cells
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Peripheral blood mononuclear cells in heparinized peripheral blood and SFMCs in heparinized joint fluid were purified using a Ficoll-Histopaque gradient (1.077 g/mL; GE Healthcare Bio-Sciences, Piscataway, NJ, USA). CD14+ monocytes were enriched from SFMCs, which was possible due to the monocytes’ ability to stick to the surface of tissue culture dishes. Briefly, SFMCs were plated on culture dishes for 1 h, then detached using 0.02% EDTA in cold phosphate-buffered saline. The purification of CD14+ monocytes was >95%, as confirmed by flow cytometry. PBMCs, SFMCs, and purified CD14+ monocytes were grown in α-minimum essential media (MEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (i.e., α-MEM complete medium, all from Life Technologies, Carlsbad, CA, USA).
RAW264.7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) containing 10% FBS and 1% penicillin/streptomycin (i.e., DMEM complete medium). For osteoclast differentiation, RAW264.7 cells were cultured in α-MEM complete medium.
RAW264.7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) containing 10% FBS and 1% penicillin/streptomycin (i.e., DMEM complete medium). For osteoclast differentiation, RAW264.7 cells were cultured in α-MEM complete medium.
5
Isolation and Characterization of CD8+ T Cell Subsets
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Peripheral blood mononuclear cells (PBMCs) in heparinized peripheral blood were isolated using a Ficoll-Histopaque gradient (1.077 g/mL; GE Healthcare Biosciences, Piscataway, NJ, USA). The following anti-human Abs were used for flow cytometry staining: allophycocyanin-anti-CD8, phycoerythrin-anti-CCR7 (all from BD Biosciences, San Jose, CA, USA), and eFluor® 450NC-anti-IL-7Rα (CD127) (eBioscience, San Diego, CA, USA).
Fluorescence-activated cell sorter (FACS) staining was performed as described (13 (link)). Staining intracellular molecules was performed after permeabilization using a Cytofix/Cytoperm kit (BD Biosciences). The stained cells were analyzed on a BD LSRII® (BD Biosciences) with FACSDiva software, and the data were analyzed using FlowJo® software (TreeStar, Ashland, OR, USA).
CD8+ T cells were enriched from PBMCs using the Human CD8+ T Cell Isolation kit (Miltenyi Biotec) and then stained with Abs and sorted into naïve (CCR7+CD45RA+), IL-7Rαhigh, and IL-7Rαlow EM (CCR7−CD45RA+/−) CD8+ T cells using a BD FACSAria® (BD Biosciences).
Fluorescence-activated cell sorter (FACS) staining was performed as described (13 (link)). Staining intracellular molecules was performed after permeabilization using a Cytofix/Cytoperm kit (BD Biosciences). The stained cells were analyzed on a BD LSRII® (BD Biosciences) with FACSDiva software, and the data were analyzed using FlowJo® software (TreeStar, Ashland, OR, USA).
CD8+ T cells were enriched from PBMCs using the Human CD8+ T Cell Isolation kit (Miltenyi Biotec) and then stained with Abs and sorted into naïve (CCR7+CD45RA+), IL-7Rαhigh, and IL-7Rαlow EM (CCR7−CD45RA+/−) CD8+ T cells using a BD FACSAria® (BD Biosciences).
6
Isolation and Characterization of Regulatory T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll Histopaque gradient (GE Health Care Bio-Sciences). CD3+ T cells were isolated by negative selection using untouched human T cell isolation kit (Life technologies, Cat# 11344D). Purity of CD3+ T cells was confirmed to be above 97%. Cells were cultured in RPMI culture media with 10% FCS, 1% Penicillin/Streptomycin, and 1% L-glutamine (all from Corning CellGro except for FCS, which was from Gibco) for 3 days, and stained with PE Cy7-conjugated anti-CD4 (Clone: SK3, Cat# 557852, RRID : AB_396897) and phycoerythrine (PE)-conjugated anti-CD25 (Clone: M-A251, Cat# 555432, RRID : AB_395826, both from BD Biosciences). The cells were permeabilized as per the manufacturer’s instructions and stained with AF-647-conjugated anti-FoxP3 (Biolegend, Clone: 150D, Cat# 320014, RRID : AB_439750).
7
Isolation and Characterization of CD8+ T Cells
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Peripheral blood mononuclear cells (PBMCs) were purified from heparinized peripheral blood using a Ficoll–Histopaque gradient (1.077 g/mL; GE Healthcare Life Sciences, Piscataway, NJ, USA) and stored in liquid nitrogen after suspension in freezing media including 50% fetal bovine serum, 10% dimethyl sulfoxide, and 40% RPMI-1640 (Invitrogen, Carlsbad, CA, USA) until further analysis30 (link). To obtain CD8+ T cells, thawed PBMCs were stained with appropriate Abs and sorted using the FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA).
Abs used for flow cytometry and sorting included Alexa 700–anti-CD8 (1:100; Cat.No. 557945; clone, RPA-T8), allophycocyanin–anti-PD-1 (1:5; Cat. No. 558694; clone, MIH4) and FITC–anti-Ki-67 (1:25; Cat. No. 51-36524X; clone, B56) (all from BD Biosciences). Staining intracellular molecules was performed after permeabilization using a Cytofix/Cytoperm kit (BD Biosciences). Stained cells were analyzed using an LSR II flow cytometer (BD Biosciences) with the FACSDiva software. Data were analyzed using the FlowJo software (TreeStar, Ashland, OR, USA).
Abs used for flow cytometry and sorting included Alexa 700–anti-CD8 (1:100; Cat.No. 557945; clone, RPA-T8), allophycocyanin–anti-PD-1 (1:5; Cat. No. 558694; clone, MIH4) and FITC–anti-Ki-67 (1:25; Cat. No. 51-36524X; clone, B56) (all from BD Biosciences). Staining intracellular molecules was performed after permeabilization using a Cytofix/Cytoperm kit (BD Biosciences). Stained cells were analyzed using an LSR II flow cytometer (BD Biosciences) with the FACSDiva software. Data were analyzed using the FlowJo software (TreeStar, Ashland, OR, USA).
8
Isolation and Characterization of PBMCs from Macaque Blood
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PBMCs were isolated from whole blood anticoagulated with heparin (132 Units per 8 ml blood) (BD Biosciences, USA) using standard methods. Of note is that the material used for density gradient centrifugation was adjusted dependent on the macaque species, with a Ficoll Histopaque gradient (GE Healthcare, USA) used with Rhesus macaque blood and a Percoll gradient (GE Healthcare, USA) used with cynomolgus macaques. PBMCs were stored at −180 °C until resuscitated for the flow cytometry assay. Samples were analysed on a BD Fortessa flow cytometer (Becton Dickinson, USA) and data was analysed using FlowJo software (FlowJo, USA). Lymphocytes and monocytes were gated using forward and side scatter characteristics. Data was collected using with method for 17 RM, 24 CCM and 23 MCM pre-infection.
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