The IFN-γ ELISPOT assay was performed as described previously27 (link). Ex vivo frequencies of CD8+ T cells responding to Mtb antigens were determined using two methods. First, CD4+ T cells were depleted using magnetic beads (Stemcell Technologies) and 250,000 CD4-depleted PBMC per well were tested in duplicate and incubated with peptide pool (5 μg/ml) overnight (CD8/others). Second, CD8+ T cells were positively selected from PBMC using magnetic beads (Stemcell Technologies) such that >97% of the cell population were CD8+ T cells. These CD8+ T cells were used as a source of responder T cells and tested in duplicate at a cell concentration of 250,000 cells per well. Autologous DC (20,000 cells/well) were used as APC and peptide pools (5 μg/ml, final concentration of each peptide) were added to the assay (CD8/DC). For assays using T cell clones, T cells (1,000, 5,000, or 10,000 cells/well) were incubated with autologous LCL or DC (20,000 cells/well) in the presence or absence of antigen or incubated with DC infected with Mtb (MOI 30:1). Negative and positive controls were included in all assays and consisted of wells containing T cells and DC either without antigen or without antigen but with inclusion of phytohemagglutanin (PHA, 10 μg/ml), respectively. For all assays, IFN-γ was assessed by ELISPOT after 18 hours of co-culture.
Magnetic beads
Manufactured by STEMCELL
Sourced in Canada, France
Magnetic beads are small, spherical particles made of a magnetic material that can be used in various laboratory applications. They are designed to be easily separated and manipulated using a magnetic field. Magnetic beads can be used for a variety of purposes, such as cell separation, protein purification, and nucleic acid isolation.
Automatically generated - may contain errors
Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (5 mentions)
Live dead kit, by Thermo Fisher Scientific (3 mentions)
Odn2006, by InvivoGen (3 mentions)
Cd14 positive selection, by STEMCELL (2 mentions)
Anti cd28 28, by Thermo Fisher Scientific (2 mentions)
Rhil 2, by Thermo Fisher Scientific (2 mentions)
Facsaria, by BD (2 mentions)
Ovalbumin (ova), by STEMCELL (2 mentions)
Ovalbumin (ova), by Merck Group (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Xf assay media, by Agilent Technologies (1 mentions)
Xf24 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Xf cell mito stress kit, by Agilent Technologies (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Cd3 immunomagnetic beads, by Miltenyi Biotec (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
Anti mouse cd3 antibody, by BioLegend (1 mentions)
Anti mouse cd28 antibody, by BioLegend (1 mentions)
Histopaque gradient, by Merck Group (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
53 protocols using magnetic beads
1
Detecting CD8+ T Cell Responses to Mycobacterial Antigens
2
Isolation and Differentiation of CD4 T Cells and DCs
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CD4 T cells and DCs were extracted from the peripheral blood of 3 healthy volunteers. PBMCs were obtained from 40 mL peripheral blood by density gradient centrifugation, resuspended in RPMI 1640 medium, and maintained at 37°C for 2 h. The suspension cells were separated by magnetic beads (Stemcell, Canada) to obtain CD4 T cells. Dynabeads Human T-activator CD3/CD28 (Miltenyi, Germany) was added to cells at a ratio of 1 : 2. Subsequently, the CD4 T cells were cultured in a 48-well plate at a density of 2 × 106 cells/mL with X-vivo complete medium. The adherent cells were stimulated with RPMI 1640 complete medium containing 1000 U/mL GM-CSF and 500 U/mL IL-4 for 6 days to obtain immature DCs, followed by stimulation with 10 ng/mL TNF-α, 1000 U/mL IL-6, 10 ng/mL IL-1β, and 1 μg/mL PGE2 for 1 day to obtain mature DCs.
3
Metabolic Profiling of Activated T Cells
4
T Cell Isolation and Enrichment
5
T-Cell Isolation and Activation Assay
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Peripheral blood mononuclear cells were isolated from freshly collected blood samples by density‐gradient centrifugation. Human total CD4+ T‐cells, naïve CD4+ T‐cells, human total CD8+ T‐cells and mouse CD4+ T‐cells were purified using magnetic beads (all from STEMCELL Technologies, Canada). Human T‐cells were cultured and stimulated with anti‐CD3/CD28 beads at a ratio of 1:2.5 for indicated days. Mouse CD4+ T‐cells were activated with anti‐mouse CD3 antibody (BioLegend, USA, 2 μg/ml) and anti‐mouse CD28 antibody (BioLegend, USA, 2 μg/ml). Purity of cell population was checked by FACS (>95%). DFX (Sigma‐Aldrich, USA), CPX (MCE, USA), DFO (Sigma‐Aldrich, USA), transferrin (TF, Sigma‐Aldrich, USA) and rapamycin (Selleck Chemicals, USA) were included in some experiments.
6
Isolation and Enrichment of NK Cells
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To isolate the PBMCs, a histopaque gradient (Sigma-Aldrich, St. Louis, MO) and SepMate tubes (Stemcell Technologies, Vancouver, Canada) were used. Adult blood was derived from healthy volunteers (Memorial Blood Center, Minneapolis, MN). To enrich the NK cells, magnetic beads (Stemcell Technologies) were used according to the manufacturer´s protocol by performing a negative selection. The purity was determined by flow cytometry. The samples were obtained after informed consent and in accordance with the University of Minnesota human subjects Institutional Review Board and the Declaration of Helsinki.
7
Isolation and Characterization of Naive CD4+ T Cells
8
Measuring Neutralizing Antibody Potency
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The TZM-bl assay was performed as described [41 (link)]. In brief, virus was added to cells in the presence of DEAE-dextran (Sigma), washed 1× on day 1 and luminescence was measured on day 2 using luciferase substrate Bright-Glo (Promega).
Human PBMC-based assay was performed as described [42 (link)]. Serially diluted SHIVIG was incubated with virus for 1 h at 37°C. The virus/SHIVIG mixture was then added to the cells. Supernatant aliquots were harvested every other day starting on day 3. To remove anti-Gag antibodies that could interfere with the p27 assay read-out, plates were washed 5 times on day 4. The levels of p27 in supernatants were assayed first in wells containing only cells plus virus. When p27 levels were in the linear phase of increase in these control wells, neutralization was assessed for test samples. To analyze the role of NK cells, the same PBMC assay was run with and without NK cells from the same donor. PBMC were depleted of NK cells using an anti-CD56 mAb linked to magnetic beads according to the manufacturer’s protocol (Stemcell Technologies).
Human PBMC-based assay was performed as described [42 (link)]. Serially diluted SHIVIG was incubated with virus for 1 h at 37°C. The virus/SHIVIG mixture was then added to the cells. Supernatant aliquots were harvested every other day starting on day 3. To remove anti-Gag antibodies that could interfere with the p27 assay read-out, plates were washed 5 times on day 4. The levels of p27 in supernatants were assayed first in wells containing only cells plus virus. When p27 levels were in the linear phase of increase in these control wells, neutralization was assessed for test samples. To analyze the role of NK cells, the same PBMC assay was run with and without NK cells from the same donor. PBMC were depleted of NK cells using an anti-CD56 mAb linked to magnetic beads according to the manufacturer’s protocol (Stemcell Technologies).
9
CD4+ T Cell Activation and Monocyte Co-culture
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PBMC were isolated by Ficoll-Hypaque density gradient centrifugation (Pharmacia LKB Biotechnology, Piscataway, NJ), from citrated (0.38%) venous blood from healthy donors obtained after informed consent, and ethical approval from the NHS Lothian Board, and conformed to the provisions of the Declaration of Helsinki. CD4+ T cells were negatively isolated using magnetic beads (StemCell, purification > 97%), Monocytes were purified by CD14+ positive selection (StemCell, purifity >90%). Cells were cultured in RPMI 1640 with 10% fetal calf serum at 1×106 per well in 48-well plate pre-coated with anti-CD3 (OKT3, 5μg/ml), anti-CD46 (MC120.6, 10 μg/ml kindly provided by Dr. Chantal Rabourdin-Combe, recognizing the SCR1 domain of CD46) or anti-CD28 (28.2, Ebiosciences, 5μg/ml). Activated primary T cells also received rhIL-2 (Life Technologies - 10U/ml). Co-cultures were performed by adding 0.2 million of monocytes followed by 0.2 million of T cells per well. For the blocking experiments, a blocking anti-CD86 (LEAF Purified anti-CD86, clone IT 2.2, 10 μg/ml, Biolegend) or irrelevant IgG1 (LEAF Purified IgG1, clone MODC21, 10 μg/ml) was added to the culture.
10
Generation of Humanized AML Xenograft Mice
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