CD4+ T cells were isolated from cryopreserved PBMC of HIV-infected patients using a magnetic negative selection kit (Stem Cell Technologies, Vancouver, Canada) following manufacturer's protocol. Purified CD4+ T cells were stained with antibodies against CD25, CD127 and CD45RA. After 30 min incubation at 4°C in the dark, cells were washed and sorted using the BD-FACSAria II (BD Biosciences).
Magnetic negative selection kit
Manufactured by STEMCELL
Sourced in Canada, United Kingdom
The Magnetic Negative Selection Kit is a tool for the isolation of specific cell types from heterogeneous cell populations. It utilizes magnetic beads coated with antibodies that bind to unwanted cells, allowing the desired cells to be isolated by magnetic separation.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 2, by BD (1 mentions)
X vivo media, by Corning (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Human ab serum, by Omega Scientific (1 mentions)
L4391 1mg, by Merck Group (1 mentions)
Pe conjugated anti mouse cd23 clone b3b4, by BioLegend (1 mentions)
Negative magnetic bead selection kit, by STEMCELL (1 mentions)
Taqman low density array, by Thermo Fisher Scientific (1 mentions)
Cxcl1, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Methyl 3h thymidine, by PerkinElmer (1 mentions)
Ckx41, by Olympus (1 mentions)
3h thymidine, by PerkinElmer (1 mentions)
12 protocols using magnetic negative selection kit
1
Isolation and Sorting of CD4+ T Cells
2
Isolating Immune Cells from Metastatic Melanoma
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Peripheral blood samples and serum were obtained from patients with metastatic melanoma under an Institutional Review Board-approved protocol.15 (link) Samples were coded with an anonymized 5-digit number and their identity was unknown to those performing the experiments. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient with 1.077 g/mL Ficoll-Paque (GE Healthcare, Chicago, Illinois, USA). For cryopreservation, PBMCs were re-suspended in human AB serum (Omega Scientific; Tarzana, California, USA) with 10% dimethyl sulfoxide (Sigma-Aldrich, St Louis, Missouri, USA), frozen at −80°C and stored in liquid nitrogen. CD3+ and CD8+ T cells were isolated using a magnetic negative selection kit (StemCell Technologies, Vancouver, British Columbia, Canada). Cells were cultured with 10% AB human serum in beta-mercaptoethanol, non-essential amino acids, HEPES, penicillin, streptomycin and gentamicin supplemented X-VIVO media (Corning, Corning, New York, USA). Serum was obtained from whole blood patient samples, centrifuged to remove cells and frozen at −80°C until time of use.
3
Isolation and NLRP3 Activation of Murine Neutrophils
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Primary neutrophils were isolated from the bone marrow of femurs of mice using the StemCell Technologies magnetic negative selection kit (Catalog # 19762) according to the manufacturer’s protocol. Femurs from at least n = 2 mice were pooled for neutrophil isolations, as we found the higher cell input improved enrichment purity. Purity was confirmed to be at least 95% (26 (link)). For NLRP3 activation, cells were treated with LPS (1 μg/ml, Sigma #L4391-1MG, strain 0111:B4) for 4 h, followed by 45 min ATP (5 mM, Sigma # A7699-1G).
4
Purification of Mouse T Cells
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The fresh spleen was collected from a healthy BALB/c mouse for each experiment, followed by mechanical digestion in sterile PBS supplemented with 2% FBS. A single-cell suspension was prepared using a 70-μm strainer, centrifuged 10 min at 300g and resuspended at 1 × 108 cells/ml. A magnetic negative selection kit was then used to purify out T cells (StemCell). Purified T cells were incubated in RPMI (10% FBS and 1% penicillin/streptomycin) at 1 × 106 cells/m per liter overnight before using in cytolysis experiment.
5
Reactivation of CD4+ T cells
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Total CD4+ T cells were isolated from cryopreserved PBMCs using magnetic negative selection kit (StemCell Technology, Vancouver, Canada). Total CD4+ T cells were exposed to GSK445A for 30 minutes, washed and cultured for a total of 18 hours in the presence of antiretroviral drugs (200 nM raltegravir, 200 nM 3TC). 162 nM PMA and 1 μg/mL ionomycin stimulation was used as a positive control for reactivation.
6
CD40-Mediated B Cell Activation
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B cells were purified from hCD40Tg/FcγR null mouse spleens by magnetic negative selection kit (StemCell Technologies, UK). Totally, 1 × 105 B cells were incubated with various anti-CD40 mAbs at 10 µg/mL in 96-well round-bottom plates for 3 days and then analysed for surface expression of CD23 using PE-conjugated anti-mouse CD23 (clone B3B4, Biolegend) by flow cytometry.
7
Murine Treg Suppression Assay
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The spleens from CD45.2 FOXP3-GFP mice were harvested 4 days after treatment with LNP. The CD4 T cells were isolated using a magnetic bead negative selection kit from StemCell. The enriched CD4 T cells were then sorted based on GFP expression. In parallel, CD45.1 conventional T cells were isolated by a magnetic negative selection kit from StemCell, and the cells were labeled with Cell Trace Violet (CTV). The CD45.2 GFP Tregs and the CTV-labeled CD45.1 conventional T cells were incubated at various ratios for 3 days in the presence of 30 K splenocytes from Rag1−/− mice and 0.3 μg/mL anti-CD3 antibody. After 3 days in culture, the cells were stained and analyzed by flow cytometry for CTV peak dilutions in the CD45.1 population.
8
Neutrophil Apoptosis Gene Expression
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Evaluation of neutrophil apoptosis-related genes was described in details and published elsewhere [28 (link)]. In brief, neutrophils were isolated from the blood of patients with GPA (active and remission) and healthy controls using magnetic negative selection kit (STEMCELL Technologies Inc., Canada) and expression of genes related to process of apoptosis was examined using the qPCR method (TaqMan Low Density Array, ThermoFisher Scientific).
9
Isolation of Naive T Cell Subsets
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Naive CD4, total CD4, or total CD8 T cells were isolated from dissociated and pooled spleen, axillary, brachial, inguinal and mesenteric lymph nodes using magnetic negative selection kits (Stemcell Technologies) as described previously (Estin et al., 2017 (link)). Naive CD4 T cells were used for naive T cell trafficking experiments. CD8 T cells were used for OT-I experiments, and islet trafficking experiments. CD4 T cells were used for all other trafficking experiments, microscopy experiments, and in vitro experiments.
10
Neutrophil Chemotaxis Assay with CXCL1
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Wells of a 24 well plate were prepared with 3 μm transwell inserts as above and with 100 ng/mL CXCL1 (Peprotech) in the bottom chamber. Neutrophils were isolated from the bone marrow of WT or FMNL1 KO mice using magnetic negative selection kits (StemCell Technologies) and then 1 × 106 cells were added to the top chambers of the transwells. Cells were allowed to migrate for 1 hr at 37°C and then quantified as above.
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