Peripheral blood sorts from the indicated subsets were performed using magnetic bead kits from Miltenyi Biotec (Bergisch Gladbach, Germany) or via antibody labeling and a FACS ARIA cell sorter (Becton Dickinson). In Group A animals, large-volume peripheral blood draws were collected immediately prior to SHIV infection, and approximately 100 days after SHIV infection. In Group B, draws were collected from infected, suppressed, transplanted animals immediately prior to cART withdrawal, and approximately 100 days after viral rebound following withdrawal of cART. Total genomic DNA was isolated from each bead-sorted sample, as well as from hemolysed total peripheral and bone marrow white blood cells (WBC, BM-WBC), Ficoll-sorted PBMC, and the granulocyte-enriched Ficoll pellet fraction (“GRANS”). Purity of bead-enriched fractions was confirmed by flow cytometry [23 (link)]. The percentage of CCR5-edited alleles in each sample was measured using Illumina MiSeq. To calculate SHIV-dependent enrichment in each subset, values during productive SHIV infection (primary infection or post-cART withdrawal viral rebound) were divided by values from pre-infection or cART-suppressed infection time points, respectively.
Magnetic bead kit
Manufactured by Miltenyi Biotec
Sourced in Germany, Canada
The Magnetic Bead Kit is a versatile tool for biomolecule separation and purification. It utilizes magnetic beads coated with specific ligands to capture and isolate target molecules from complex samples. The core function of this product is to facilitate efficient and reproducible separation of biomolecules, such as proteins, nucleic acids, or cells, from a variety of biological matrices.
Automatically generated - may contain errors
Lab products found in correlation
Mouse b cell isolation kit, by STEMCELL (2 mentions)
Penicillin streptomycin antibiotic, by Thermo Fisher Scientific (2 mentions)
2 β mercaptoethanol, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Facsaria cell sorter, by BD (1 mentions)
Facsaria 2 machine, by BD (1 mentions)
Anti cd19 j3 119, by Beckman Coulter (1 mentions)
Anti cd8 b9.11, by Beckman Coulter (1 mentions)
Anti cd38 at 1, by STEMCELL (1 mentions)
Cd95 dx2, by BD (1 mentions)
Mouse t cell isolation kit, by STEMCELL (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Foxp3 staining buffer kit, by Thermo Fisher Scientific (1 mentions)
Facsuite software, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Heat inactivated, by Atlanta Biologicals (1 mentions)
6 protocols using magnetic bead kit
1
Quantifying CCR5-Edited Alleles in Subsets
2
Lymph Node and GI Tissue Analysis
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At the indicated time points, inguinal lymph nodes were collected and flash frozen. GI biopsies from upper GI (duodenum) and lower GI (colon) were collected as described previously.29 (link) Single-cell suspensions were prepared from cell culture or peripheral tissues. Peripheral blood cell subsets were sorted using magnetic bead kits from Miltenyi Biotec (Bergisch Gladbach, Germany) or through antibody labeling and a FACSAria II machine (BD Biosciences, Franklin Lakes, NJ, USA). The following antibodies (clones) were purchased from BD Biosciences: CD3 (SP34-2), CD4 (L200), CD45 (D058-1283), CCR5 (3A9), CD11b (ICRF44), CD14 (M5E2), CD16 (3G8), CD34 (563), CD28 (CD28.2), and CD95 (DX2). Anti-CD8 (B9.11) and anti-CD19 (J3-119) were purchased from Beckman Coulter (Brea, CA, USA). Anti-CD38 (AT-1) was purchased from STEMCELL Technologies (Vancouver, BC, Canada). For surface marker measurement, individual antibodies were added to the cell suspension alone or together. Isotype control antibodies were used for gating the positive group of target cells. Cells were stained with antibodies for 20 min at room temperature followed by washing twice with PBS plus 2% FBS. After resuspension, cells were subjected to FACS analysis in less than 8 h.
3
Splenic B and T Cell Purification
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Splenic B cells were purified by negative selection as previously reported4 (link). Briefly, B cells were isolated by anti-CD43 Ab-mediated negative selection, using a magnetic bead kit (Miltenyi Biotec, Auburn, CA) or mouse B cell isolation kit (STEMCELL, Vancouver, Canada), according to the manufacturers’ protocols. Mouse splenic pan-T cells were purified by negative selection with a mouse T cell isolation kit (STEMCELL). Cells, were maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10 μM 2-β-mercaptoethanol (Sigma Aldrich, St. Louis, MO), 10% heat-inactivated FBS (Atlanta Biologicals, Atlanta, GA, USA), 2 mM L-glutamine (Life Technologies), and 100 U/ml penicillin-streptomycin antibiotics (Life Technologies). This is referred to as B cell medium (BCM10).
4
Isolation and Analysis of Murine Naive CD8+ T Cells
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CD8+ T cells from spleens of mice were enriched by positive selection using a magnetic bead kit (Miltenyi Biotec Inc., Bergisch Gladbach, Germany) prior to activation. CD62LhiCD44loCD8+ were sorted (FACSAria (BD Bioscience, San Jose, CA, USA) and used as naïve CD8+ T cells (purity ≥ 99%) throughout the experiment. through service provided by Flow Cytometric Analyzing and Sorting Core Facility (First Core Laboratory, NTU, College of Medicine).
To determine the expression of surface antigens, live cells were stained by indicated fluorochrome-conjugated antibodies for 20–30 min on ice. Detection of intracellular cytokines was performed after 6-h stimulation with phorbol myristate acetate (PMA, 10 ng/mL)/Ionomycin (1 µg/mL) and 4-h culture in Brefeldin A (10 µg/mL). Live cells were fixed and permeabilized with cytofix-cytoperm kit (BD Biosciences) for staining of intracellular proteins. Intracellular Foxp3 was detected by Alexa 488 anti-Foxp3 (clone FJK-16s, eBioscience) after fixation and permeabilization using a Foxp3 staining buffer kit (eBioscience). BD FACSVerse™ (BD Biosciences) was employed for cell acquisition and BD FACSuite™ software for data analysis.
To determine the expression of surface antigens, live cells were stained by indicated fluorochrome-conjugated antibodies for 20–30 min on ice. Detection of intracellular cytokines was performed after 6-h stimulation with phorbol myristate acetate (PMA, 10 ng/mL)/Ionomycin (1 µg/mL) and 4-h culture in Brefeldin A (10 µg/mL). Live cells were fixed and permeabilized with cytofix-cytoperm kit (BD Biosciences) for staining of intracellular proteins. Intracellular Foxp3 was detected by Alexa 488 anti-Foxp3 (clone FJK-16s, eBioscience) after fixation and permeabilization using a Foxp3 staining buffer kit (eBioscience). BD FACSVerse™ (BD Biosciences) was employed for cell acquisition and BD FACSuite™ software for data analysis.
5
Isolation and Culturing of Splenic B and T Cells
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Splenic B cells were isolated by negative selection as previously reported2 (link). Briefly, splenic B cells were isolated by anti-CD43 Ab-mediated negative selection, using a magnetic bead kit (Miltenyi Biotec, Auburn, CA) or mouse B cell isolation kit (STEMCELL, Vancouver, Canada), according to manufacturers’ protocols. Splenic T cells were isolated using a T cell isolation kit (STEMCELL), following the manufacturer’s protocol. Cells, including cell lines, were maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10 μM 2-β-mercaptoethanol (Sigma), 10% heat-inactivated FCS (Atlanta Biologicals, Atlanta, GA, USA), 2 mM l-Glutamine (Life Technologies), and 100 U/ml of penicillin-streptomycin antibiotics (Life Technologies). In glucose deprivation assays, cells were washed in sterile phosphate buffered saline (PBS, Life Technologies) to remove residual glucose-containing medium and incubated in glucose free RPMI 1640 medium supplemented by glutamine and 1 mM sodium pyruvate (Life Technologies) and 0.1% bovine serum albumin (BSA).
6
Regulatory T Cell Suppression Assay
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BMDCs were cultured in the presence or absence of IKK inhibitor and loaded with ovalbumin overnight. Ovalbumin-loaded BMDCs were co-cultured with naive Thy1.1+ OT-II T cells as described above. After 5 d, T cells were collected, and CD25+ cells were purified by cell sorting. Naive polyclonal CD4+ T cells were isolated from wild-type C56BL6/J mice using a magnetic bead kit (Miltenyi Biotec) and labeled with CellTrace Violet as described above. Labeled B6 T cells were added to 96-well plates at 5 × 104 cells per well in 100 µl RPMI. Purified in vitro generated Treg were added at ratios of 1:16 to 1:1 in 100 µl RPMI. Anti-CD3 and anti-CD28 antibodies were added to a final concentration of 5 and 1 µg/ml, respectively. After 3 d, T cells were collected and Thy1.1− T cell proliferation was analyzed by flow cytometry.
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