Mouse bone marrow-derived macrophages were isolated from femurs and matured for six days with macrophage colony-stimulating factor (M-CSF) containing L929 cell supernatant supplementation as described [46 (link)]. Human macrophages were matured under presence of 20 ng/ml recombinant human M-CSF (Miltenyi Biotec) for eight days from PBMC-derived monocytes sorted for CD14 expression by positive magnetic bead selection (Miltenyi Biotec) with purity >95%. Fresh buffy coats for PBMC isolation by gradient centrifugation (PAA) were obtained from the German Red Cross. For co-culture experiments, ABCB5+-derived MSCs or donor-matched ABCB5− HDFs were plated to adhere at 2×104 cells/well in 24-well plates in 0.5 ml DMEM with 10% high quality fetal bovine serum, 100 U/ml penicillin / streptomycin and 2 mM L-glutamine. After 24 h macrophages were seeded on top at 1×105 cells/-well in 0.5 ml, resulting in a 1:5 cell ratio unless indicated differently. Co-cultures were incubated with 50 U ml−1 recombinant mouse or human IFN-γ (R&D Systems) for 24 h and then stimulated with 20 ng ml−1 LPS (Sigma-Aldrich) and 50 U ml−1 IFN-γ for another 24 h period before supernatants were harvested and analyzed by ELISA (R&D Systems).
Magnetic bead positive selection
Manufactured by Miltenyi Biotec
Sourced in Germany
Magnetic bead positive selection is a lab equipment product used for the isolation and purification of specific cell populations from complex biological samples. The core function of this product is to utilize magnetic beads coated with antibodies or ligands that can bind to and capture the target cells of interest, allowing for their efficient separation and enrichment from the sample.
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Lab products found in correlation
Immobilon p, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
3 amino 9 ethylcarbazole, by Merck Group (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Anti ifn γ mab clone an18, by Mabtech (3 mentions)
Biotinylated anti mouse ifn γ mab r4 6a2, by Mabtech (3 mentions)
Recombinant human il 2, by Merck Group (2 mentions)
Dynabeads mouse t activator cd3 cd28, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
40 m cell strainer, by Avantor (2 mentions)
Recombinant human m csf, by Miltenyi Biotec (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
γδ t cell isolation kit, by Miltenyi Biotec (1 mentions)
Lymphocyte separation medium, by Corning (1 mentions)
Facscalibur, by BD (1 mentions)
Negative magnetic bead selection, by STEMCELL (1 mentions)
Aria flow cytometer, by BD (1 mentions)
15 protocols using magnetic bead positive selection
1
Macrophage Co-Culture Activation Assay
2
Isolation and RNA Extraction from CD4+ T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood by standard Ficoll-Hypaque procedures. CD4+ T cells were enriched by positive magnetic-bead selection (Miltenyi, Gladbach, Germany) according to the manufacturer’s instructions. RNA was extracted from CD4+ T cells using TRIzol (Invitrogen).
3
Isolation and Activation of Mouse CD8+ T Cells
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Spleens were harvested, mashed over a 40-µm cell strainer (VWR, 10199 – 654 ), and CD8+ T-cells purified by positive magnetic bead selection (Miltenyi Biotec, 130-117-044 ) according to manufacturer’s instructions. Purified CD8+ T-cells were counted and cell diameter measured using a Moxi Z mini automated counter (Orflo, MXZ001 ). The cells were characterized using Flow cytometry described below or plated at 1 × 106 (24-well plate) or 5 × 105 (48-well plate) and activated with Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher, 11456D ) in a 1:1 beat to T-cell ratio and cultured in 2 ml (24WP) or 1 ml (48WP) RPMI 1640 (Thermo Fisher, 21875 ) supplemented with 10% Fetal Bovine Serum, (Thermo Fisher, 10270 – 106 ), 50 µM 2-mercaptoethanol (Thermo Fisher, 21985023 ), 10 U/ml penicillin-streptomycin (Thermo Fisher, 15140122 ), and 10 U/ml recombinant human IL-2 (Sigma, 11011456001 ), and incubated at 37 °C for 3 days in a humidified CO2 incubator.
4
Isolation of PBMC and T Cell Subsets
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Peripheral blood mononuclear cells (PBMC) were obtained from a buffy coat, or whole blood was isolated by standard density gradient separation using Lymphocyte Separation Medium (Corning). Monocytes were isolated from PBMC by positive magnetic bead selection (Miltenyi Biotec), and CD4+ T cells and γδ T cell subsets (refers to Vδ1 and Vδ2 T cells) were isolated by negative selection (EasySep CD4 T cell, Cat #-17952 and γδ T cell isolation kit, Cat #- 19255) according to the manufacturer’s specifications, and the differentially isolated cells were cultured or cryopreserved until use
5
Purification and Activation of Murine CD8+ T Cells
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Spleens were harvested from 8-12 week old C57BL/6J (RRID : IMSR_JAX:000664) mice, mashed over a 40 µm cell strainer (VWR, 10199-654), and CD8+ T cells were purified by positive magnetic bead selection (Miltenyi Biotec, 130-117-044) according to manufacturer’s instructions. Purified CD8+ T cells were counted and cell diameter measured using a Moxi Z mini (Orflo, MXZ001) or a TC20 (Bio-Rad, 145-0101) automated counter. 1x106 (24-well plate) or 5x105 (48-well plate) CD8+ T cells were activated with Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher, 11456D) in a 1:1 bead to T cell ratio and cultured in 2 ml (24-well plate) or 1 ml (48-well plate) RPMI 1640 (Thermo Fisher, 21875) supplemented with 10% Fetal Bovine Serum (Thermo Fisher, 10270-106), 50 µM 2-mercaptoethanol (Thermo Fisher, 21985023), 100 U/ml penicillin-streptomycin (Thermo Fisher, 15140122), and 10 U/ml recombinant human IL-2 (Sigma, 11011456001), and incubated at 37˚ C for 3 days in a humidified CO2 incubator.
6
Evaluating Bone Marrow Cell Viability and Apoptosis
7
Quantifying Virus-Specific CD8+ T Cell Responses
8
Isolation and Co-culture of B Cell Subsets and T Cells
9
Imaging Flow Cytometry of NF-κB Activation
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HEK293 cells were transiently transfected for 24 hr with HOIL-1, V5-His-HOIP, Sharpin, and/or UBE2L3. Cells were detached with TrypLE (Life Technologies), stimulated with TNF for 30 min, fixed with BD cytofix, permeabilized with 0.1% Triton X-100, and stained with rabbit anti-p65 (Santa Cruz), PE-conjugated anti-UBE2L3, and anti-V5-AlexaFluor647. Cells were washed, incubated with AlexaFluor488 F(ab)2 donkey anti-rabbit IgG (Jackson), washed, stained with DAPI. 20,000 cells per condition were acquired on Imagestream X imaging flow cytometer (Amnis). For ex vivo cell analysis, PBMCs were isolated via Histopaque from blood samples from previously genotyped healthy individuals (TwinsUK). CD19+ B cells were isolated by magnetic bead positive selection (Miltenyi) and CD14+ monocytes were isolated by negative selection (Miltenyi). Endotoxin-free MACS buffer was used throughout. Cell purity was confirmed by flow cytometry. B cells and monocytes were cultured in RPMI and stimulated with 0.1 μg/ml CD40L (Enzo) and 10 ng/ml TNF (Axxora), respectively, for up to 60 min. Cells were fixed and stained for p65 as above, washed, and stained with DRAQ5 (eBioscience), and 15,000–20,000 events per sample were acquired by Imagestream X. Data analysis was entirely automated with IDEAS software batch function applied to the entire cohort and performed fully blinded to genotype.
10
Quantification of Rhesus sjTREC in CD4+ and CD8+ Cells
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CD4+ and CD8+ cells were isolated from cryopreserved PBMC using magnetic-bead positive selection (Miltenyi Biotec). Cells were counted and then lysed in proteinase K (Roche). sjTREC in lysates and serially diluted, quantified NHP sjTREC standard plasmid27 (link) were amplified using Platinum Taq (Invitrogen) with rhesus-specific sjTREC primers and probes27 (link) using a CFX Connect or CFX96 RT-PCR instrument (Bio-Rad). Lysate sjTREC content was extrapolated from the sjTREC standard plasmid results.
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