C57BL/6 mice were immunized with the MOG35–55 peptide (200 µg/mouse, s.c.) in a CFA emulsion at the first day (Day 0). Twelve days after the immunization (Day 12), splenic CD4+ T cells were then isolated by using MACS beads (STEMCELL Technologies, Vancouver, Canada) and cultured in RPMI 1640 medium supplemented with 1% FBS and 25 µg/mL I-Ab MOG35–55 peptide (Medical and Biological Laboratories, Japan) at 37°C for 3 days. The cells were washed twice with PBS and then stained with I-Ab MOG35-55 tetramer-PE (10 μg/mL in final concentration) (Medical and Biological Laboratories, Japan) and CD4-APC-Cy7 (5 μg/mL in final concentration) (Biolegend, San Diego, CA, USA) for 1 h at 4°C in PBS containing FBS (2.5%) for analysis by flow cytometry. MOG35–55-specific CD4+ T cells were defined as I-Ab MOG35–55 tetramer+ CD4+ cells by flow cytometry analysis.
Macs beads
Manufactured by STEMCELL
Sourced in Canada
MACS Beads are magnetic beads used for cell separation in research applications. They are designed to bind to specific cell surface markers, allowing the target cells to be isolated from a heterogeneous cell population using a magnetic field.
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Lab products found in correlation
Cd4 apc cy7, by BioLegend (1 mentions)
Lsrfortessa system, by BD (1 mentions)
Sp5 laser confocal microscope, by Leica (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Anti cd40, by BD (1 mentions)
Goat anti mouse igm f ab 2, by Jackson ImmunoResearch (1 mentions)
Recombinant baff, by R&D Systems (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
Lps from escherichia coli o111 b4, by Merck Group (1 mentions)
Facsaria 3, by BD (1 mentions)
6 protocols using macs beads
1
MOG35-55 Peptide Immunization and CD4+ T Cell Analysis
2
Quantifying Colonic Cell Apoptosis
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Colonic epithelial cells were isolated as previously described,39 (link) and colonic CD4+ and CD8+ T cells as well as CD11c+ dendritic cells were further isolated from laminar propria cells using magnetic-activated cell sorting (MACS) beads (STEMCELL Technologies, Vancouver, Canada). Isolated cells were treated with various concentrations of TRAIL for 24 h at 37 °C. To detect apoptotic cells, cytoplasmic histone-associated DNA fragments from each treatment group were measured with the Cell Death Detection ELISA PLUS system according to the manufacturer’s protocol (Roche Mannheim Biochemicals, Mannheim, Germany). In brief, cells were lysed, and lysates were collected and mixed with an immunoreagent for 2 h, followed by reaction with a substrate solution in the dark until color had developed. The reaction was quantified using spectrophotometry at 405 nm.
3
Quantification of Immunological Synapse Formation
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Naïve CD8 T cells were isolated from control or mDia1/3 cDKO OT-I mice using a negative selection MACS purification kit (130-096-543, Miltenyi Biotec). B cells from WT mice were isolated using MACS beads (18954, Stemcell Technologies), incubated with SL8 peptide (1 μg/ml) (Sigma) for 30 min at 37°C, and then surface-stained with phycoerythrin (PE)–conjugated anti-B220 antibody (12-0452-83, eBioscience). For cell conjugation assays, equal volumes of naïve CD8 OT-I T cells and SL8 peptide–pulsed B cells were mixed and incubated at 37°C for 15 min. Conjugated cells were then fixed with 0.5% PFA for FACS analysis using the LSR Fortessa System (BD Biosciences). The percentage of conjugates within the culture was analyzed using FlowJo software (Tree Star Inc.). For imaging of the F-actin ring, cell conjugates were fixed with 4% PFA, permeabilized, stained with Alexa Fluor 488-conjugated phalloidin, and then imaged with a Leica SP5 laser confocal microscope equipped with 100× NA 1.4 HCX PL APO CS objective lens (Leica). 3D reconstruction of stacked confocal images and en face view images were generated by Volocity software (PerkinElmer). Surface plots of 3D reconstructed images were generated using ImageJ software (NIH). Quantification of IS F-actin intensity and IS diameter was performed using ImageJ software (NIH).
4
In vitro Splenic B Cell Proliferation Assay
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Splenic B cells were isolated with MACS beads by negative selection according to the manufacturer’s protocol (STEMCELL Technologies). The purity was routinely >96% as assessed by staining with anti-B220. For the in vitro proliferation assay, splenic B cells were labeled with 1 mM CFSE (Sigma-Aldrich) for 10 min in phosphate-buffered saline (PBS) and washed twice with RPMI 1640 medium containing 10% fetal bovine serum (FBS) before plating. B cells at a density of 1 × 106 cells/ml were then cultured in normal culture medium and stimulated with F(ab’)2 goat anti-mouse IgM (10 µg/ml; Jackson ImmunoResearch), LPS from Escherichia coli O111:B4 (2 µg/ml; Sigma-Aldrich), recombinant BAFF (100 ng/ml; R&D Systems) and anti-CD40 (1 µg/ml; BD Biosciences). Normal culture medium for B cells was 1640 medium with 10% (vol/vol) FBS, 0.05 mM β-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 µg/ml streptomycin. CFSE dilution was analyzed by flow cytometry after 72 h of stimulation. In some experiments, 20 μM DFO or 100 μM FAC was applied to the cell culture medium. For the [3H]thymidine incorporation assay, purified B cells were also plated at a density of 2 × 105 cells per well (200 µl in 96-well plates) with various levels of stimulation and cultured for 72 h with the addition of 0.4 mCi [3H]thymidine for the final 12 h of culture.
5
Isolation and Sorting of Tumor-Infiltrating Lymphocytes
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CD45+ TIL or CD8+ TIL for CYTOF, bulk-RNA-seq, flow cytometry or 3D HYGTIC experiments were sorted by MACS beads (Stem cell) or by flow cytometry sorting, after confirming its purity by flow cytometric analysis. Cells (diluted in FACS) density was counted by hemacytometer manually. ≥ 10x106 cells were centrifuged, and then resuspended with specific antibodies (Supplementary Table S2
6
Investigating TRAIL-Mediated T Cell Responses
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Splenic CD4+/CD25− T cells from WT or TRAIL-R KO mice were isolated using MACS beads (STEMCELL Technologies). Isolated cells were treated with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) Abs in the presence or absence of TRAIL (10 µg/ml) for 24 h at 37 °C. Subsequently, 5 × 105 cells were transferred into Rag1 KO mice via an intraperitoneal injection. Body weight loss and clinical symptoms were recorded daily.
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