PBMC were isolated using density gradient lymphoprep (STEMCELL Technologies, USA) according to manufacturer’s instruction. Cell subpopulations, CD4+ T cells, were isolated from PBMC with CD4+ T cell isolation kit II human of Miltenyi Biotec (Miltenyi Biotec, Bergisch Gladbach, Germany), with purity of more than 95 percent [18 (link)] and according to manufacturer’s instruction.
Lymphoprep density gradient
Manufactured by STEMCELL
Sourced in Canada, United States
Lymphoprep is a density gradient medium used for the isolation of mononuclear cells from whole blood or other body fluids. It is a sterile, pyrogen-free solution of sodium diatrizoate and polysaccharide, which facilitates the separation of peripheral blood mononuclear cells by density gradient centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
True stain monocyte blocker, by BioLegend (2 mentions)
Brilliant stain buffer, by BD (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Red blood cell lysis buffer, by Thermo Fisher Scientific (2 mentions)
Zombie yellow fixable viability kit, by BioLegend (2 mentions)
Anti cd16 32, by BioLegend (2 mentions)
Collagenase type 4, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Ack buffer, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Mojosort human nk cell isolation kit, by BioLegend (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Facs lsrii flow cytometer, by BD (1 mentions)
Facsdiva acquisition software, by BD (1 mentions)
Flowlogic fcs analysis software, by Inivai Technologies (1 mentions)
70 μm sieve, by BD (1 mentions)
23 protocols using lymphoprep density gradient
1
PBMC Isolation and CD4+ T Cell Purification
2
Isolation and Characterization of CD4+ T-cells
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The obtained blood samples were immediately placed on ice and transferred to the laboratory for analysis. We used a two-step process to separate the CD4+ T-cells from the whole blood. In the first step, PBMCs were isolated by density gradient lymphoprep (STEMCELL Technologies, USA) according to the manufacturer’s instructions. In the second step, CD4+T-cells were isolated on PBMC by magneticactivated cell sorting (MACS) with a CD4+T-cell isolation Kit II human (Miltenyi Biotec, Germany) based on the manufacturer’s protocol. This kit is an indirect magnetic labeling system for the isolation of untouched CD4+Thelper cells from human PBMC by elimination of cells that contain CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ/δ, and glycophorin A with a purity of greater than 95%. Total RNA was extracted with the TRizol® reagent (Invitrogen, USA) from CD4+ T-cells according to the manufacturer’s protocol. The quantity and quality of extracted RNA were verified according to the ratio of absorbance at a 260/280 nm as measured by a NanoDrop spectrophotometer (Nanodrop 1000, Thermo Scientific, USA), and by electrophoresis on a 1% agarose gel. Total RNA samples were treated with RNA-free DNase (Fermentas, Ukraine) in order to eliminate trace amounts of contaminated DNA prior to real-time quantitative polymerase chain reaction (qRT-PCR) analysis.
3
Isolation of Peripheral Blood Mononuclear Cells
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Twelve healthy laboratory control subjects (aged 25‐35 years) were recruited from the Department of Ophthalmology, Post Graduate Institute of Medical Education and Research (PGIMER) Chandigarh, after prior informed consent. The study was duly approved by PGIMER Chandigarh institute ethics committee (No's PGI/IEC/2011/532‐533/24/11/11 and NK/1385/PhD/2484/07/09/17). This study was executed in accordance with the recommendations of the Institutional ethics committee of PGIMER. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Fifteen millilitres of peripheral blood was withdrawn from all subjects participating in the study and used for isolation of peripheral blood mononuclear cells (PBMCs) using lymphoprep™ density gradient (Stem cell technologies, Vancouver, Canada).
4
Peripheral Blood Mononuclear Cell Isolation
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Healthy donors 18 years old and older were recruited for this study under the University of Utah Institutional Review Board (IRB) protocol 67637. Written informed consent was obtained from all donors. Whole blood was obtained by peripheral phlebotomy, and peripheral blood mononuclear cells (PBMC) were isolated using a Lymphoprep density gradient (Stemcell Technologies).
5
NK Cell Isolation and Activation
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NK cells were isolated and activated as previously described [24 (link)]. Briefly, PBMCs derived from the patient or healthy donors by Lymphoprep™ density gradient (STEMCELL™ Technologies). NK cells were isolated by StemSep™ Human NK Cell Enrichment Kit. Following isolation, NK cells were incubated with; 6000RAD irradiated 5*106 PBMCs from two donors and 5*105 RPMI-8866 cells in presence of 400u/ml recombinant human IL-2 (PeproTech 200-02).
6
Isolation and Culture of Human NK Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy, anonymous donors provided by the Luxembourg Red Cross. Upon receipt, buffy coats were diluted ten times with Ca2+/Mg2+ free phosphate buffered saline (PBS) and the low-density PBMC fraction was isolated by centrifugation over a Lymphoprep density gradient (Stemcell Technologies, cat. # 07861). After centrifugation, the PBMC layer was collected, washed several times with Ca2+/Mg2+ free PBS and red blood cells were lysed with ACK buffer (ThermoFisher Scientific, cat. # A1049201). Following erythrocytes lysis, cells were washed once with Ca2+/Mg2+ free PBS, counted with Trypan blue and cell concentration adjusted for NK cell isolation. NK cells were isolated with the MojoSort human NK cell isolation kit (BioLegend, cat. # 480054) combined with a LS column (Miltenyi Biotec, cat. # 130-042-401). Isolated NK cells were cultured overnight in RPMI 1640 supplemented with 10% FBS, 10 mM HEPES (ThermoFisher Scientific, cat. # 15630056), 100 U/mL penicillin, 0.1 mg/mL streptomycin, 100 U/mL recombinant human interleukin-2 (IL-2; Peprotech, cat. # 200-02) and 10 ng/mL recombinant human IL-15 (IL-2; Peprotech, cat. # 200-15).
7
Multicolor Flow Cytometry Analysis of T Cell Subsets
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Peripheral blood mononuclear cells were isolated from heparinized venous blood (collected at baseline) over a Lymphoprep density gradient (StemCell Technologies, France) and cryopreserved in liquid nitrogen in heat-inactivated human bovine serum supplemented with 10% dimethyl sulfoxide until batch testing. After thawing, the peripheral blood mononuclear cells were stained with fluorescently conjugated mouse antihuman monoclonal antibodies (Supplemental Table S4) for multicolor flow cytometric analysis acquired on BD FACS Canto II cytometer (BD Biosciences, USA) and analyzed with FlowJo software, version 10.6.2 (BD Biosciences, USA). T cells were defined as CD45+CD3+, helper T cells as CD45+CD3+CD4+CD8−, regulatory T cells as CD45+CD3+CD4+CD8−CD25highCD127lowFoxP3+, and cytotoxic T cells as CD45+CD3+CD4−CD8+.
8
Ferret Immune Cell Isolation and Phenotyping
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Spleen, thymus and lymph node were excised from euthanized ferrets, cleaned of any connective tissue and mechanically digested in cold FACS buffer (2% (v/v) FCS, 0.02% (v/v) NaN3 in PBS) to produce a single suspension. Mechanical digestion was achieved by pressing the tissue through a 70 μm sieve (BD Biosciences) using the plunger of a 10 mL syringe. Red blood cells were removed from the spleen by Lymphoprep density gradient (Stemcell Technologies) centrifugation at 1000g for 20 min with no brake. One million cells were stained with 0.1 μg of anti-ferret CD4, washed in FACS buffer and resuspended in 150 μL of FACS buffer. Additional antibodies used for phenotyping included CD8 (OKT8), CD81 (JS081), CD11b (M1/70) (BD Biosciences), MHC class II (CAT82a) (Kingfisher) and anti-ferret Ig (H + L) (Rockland). Where required an anti-IgG2a or anti-IgG1 secondaries were used (Invitrogen). For fixation cells were resuspended in 200 μL of 4% paraformaldehyde (PFA) in PBS. Data was acquired using a BD FACS LSRII flow cytometer (BD Biosciences) equipped with 405, 488, and 633 nm excitation lasers in conjunction with FACS Diva acquisition software (BD Biosciences). Data analysis was performed using FlowLogic FCS analysis software (Inivai Technologies).
9
Multiparametric Phenotyping of PBMCs
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PBMCs were isolated from peripheral blood using a Lymphoprep density gradient (STEMCELL Technologies, Vancouver, Canada). Each sample received a cocktail containing 10 μL Brilliant Stain Buffer (BD Biosciences, Franklin Lakes, NJ, USA), 5 μL True-Stain Monocyte Blocker (BioLegend, San Diego, CA, USA), and the following surface marker antibodies: anti-CD19 (PE-Dazzle594), anti-CD3 (APC), anti-CD16 (Alexa700), HLA-DR (APC/Fire750), and anti-CTLA-4 (PE-Cy7). The following antibodies were then added to each tube individually: anti-CD8 (BUV496), anti-CD4 (BUV661), anti-CD45 (BUV805), anti-CD103 (BV421), anti-TIM3 (BV605), anti CD56 (BV650), anti-LAG-3 (BV711), anti-CD14 (BB785), and anti-PD-1 (BB700), before incubating at room temperature for 30 min. Cells were fixed and permeabilized in 1× incellMAX (IncellDx, San Carlos, CA, USA) for 60 min at room temperature in the dark. Cells were washed once with 2% bovine serum albumin (BSA) solution, and analyzed using a Cytoflex LX system with 355 nm (20 mW), 405 nm (80 mW), 488 nm (50 mW), 561 nm (30 mW), 638 nm (50 mW), and 808 nm (60 mW) lasers (Beckman Coulter Life Sciences, Indianapolis, IN, USA). Analysis was performed with Kaluza version 2.1 software. The panel used in this study is shown in Supplementary Table 1.
10
Multiparametric Immune Cell Profiling
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Peripheral blood mononuclear cells were isolated from peripheral blood using Lymphoprep density gradient (STEMCELL Technologies, Vancouver, Canada). Aliquots of 200,000 cells were frozen in media that contained 90% fetal bovine serum (HyClone, Logan, UT) and 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) and stored at -70°C for <1 week to preserve viability and run as a single batch to avoid intrarun variability. Cells were blocked with 2% BSA solution for 5 min. at RT. A cocktail containing the following antibodies and reagents was added: Brilliant Stain Buffer (BD Biosciences, San Jose, CA), True-Stain Monocyte Blocker, anti-CD19-PE-Dazzle594, anti-CTLA-4 PE-Cy7, anti-CD3-APC, anti-CD16-Alexa Fluor 700 (all BioLegend, San Diego, CA), and anti-SARS-CoV-2 Spike S1 Subunit-Alexa Fluor 405 (R&D Systems, Minneapolis, MN). The following antibodies were then added: anti-CD8 BUV496, anti-CD4-BUV661, anti-CD45-BUV805, anti-PD-1-BB700 (all BD Biosciences, San Jose, CA), anti-CD56-BV711 (BioLegend, San Diego, CA), and anti-CD14 BV786 (BioLegend, San Diego, CA). Cells were stained for 30 min. at RT, and then washed twice with 2%BSA. Cells were fixed for 1 hour at RT in 1 mL incellMAX (IncellDx, San Carlos, CA), and then incubated with anti-FoxP3-PE antibody (BD Biosciences, San Jose, CA) for 30 min. Cells were washed twice with 2%BSA and then acquired on a 5-laser CytoFLEX LX.
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