Spleen and lymph node cells or blood cell were harvested and stained with p:I-Ab tetramers, and in a subset of experiments with CXCR5 (L138D7; BioLegend) antibody for one hour at room temperature, then incubated with anti-PE and anti-APC magnetic beads. Samples then passed over magnetized columns (Miltenyi). Bound cells were eluted from the columns and stained for 30 minutes on ice with antibodies from eBioscience to CD3e (145-2C11), CD4 (GK1.5; BD), CD8 (53-6.7), CD90.1 (HIS51;), CD90.2 (53-2.1), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD44 (IM7), CD45.2 (104), B220 (RA3-6B2), and CD45.1 (A20). Cellular viability by GhostDye Red 780 (Tonbo). Stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) for one hour at room temperature and stained overnight at 4 °C with antibodies to T-bet (4B10; BioLegend), FoxP3 (FJK-16s; eBioscience), RORγt (Q31-378; BD), and/ or Bcl-6 (K112-91; BD). Cells were analyzed on a Fortessa X-20 (BD) using FlowJo software v10 (TreeStar). The total number of tetramer-positive cells in the enriched fraction from each mouse was determined using AccuCheck Counting Beads (ThermoFisher).
Magnetized column
Manufactured by Miltenyi Biotec
Sourced in Canada
Magnetized columns are specialized laboratory equipment used for the separation and isolation of magnetically labeled cells or particles. These columns contain a matrix of ferromagnetic material that creates a strong magnetic field, allowing for the efficient capture and separation of target cells or molecules that have been labeled with magnetic particles. The core function of magnetized columns is to enable precise and gentle cell separation, a critical process in various biotechnology and medical research applications.
Automatically generated - may contain errors
Lab products found in correlation
Countbright counting beads, by Thermo Fisher Scientific (2 mentions)
Anti pe and anti apc beads, by Miltenyi Biotec (2 mentions)
T bet 4b10, by BioLegend (1 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions)
Rorγt q31 378, by BD (1 mentions)
Ghost dye red 780, by Cytek Biosciences (1 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Fortessa x20, by BD (1 mentions)
Anti pe and anti apc microbeads, by Miltenyi Biotec (1 mentions)
Cd11b beads, by Miltenyi Biotec (1 mentions)
7900ht real time machine, by Thermo Fisher Scientific (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Duoset kit, by R&D Systems (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Stemsep, by STEMCELL (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Odyssey fc scanner, by LI COR (1 mentions)
8 protocols using magnetized column
1
Tetramer-Based Immune Cell Profiling
2
Tetramer-based Enrichment of Antigen-specific Cells
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Tetramer enrichment was used to isolate antigen-specific cells from pre-immune or acutely challenged mice. A modification of the method used by Obar et al., 2008 (link) was employed. Following digestion with collagenase D, single-cell suspensions were prepared from the spleens (acutely challenged mice) or spleen and cervical, axillary, brachial, inguinal, and mesenteric lymph nodes (pre-immune mice). When possible, the same tetramer (Trp2180–188/Kb) was used in both APC and PE to ensure specificity. Anti-PE and anti-APC beads and magnetized columns (both from Miltenyi Biotec) were used to enrich for tetramer-bound cells. Samples were stained and analyzed by flow cytometry; CountBright counting beads (Invitrogen) were used for enumeration.
3
Tet-labeled Cell Enrichment Protocol
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Tet–labeled cells were enriched using anti-PE and anti-APC microbeads and magnetized columns (Miltenyi Biotech) as described previously (33 (link)). For details, see SI Appendix .
4
Comprehensive Skin Cell Characterization
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RNA was extracted using PureLink Plus columns and converted to cDNA using the High Capacity RNA-to-cDNA kit (Life Technologies). qPCR analysis was undertaken using SYBR-green PerfeCTa (Quanta) and TLDAs were used as per manufacturer’s instructions on a 7900HT Real time machine (Applied Biosystems). ELISAs were undertaken using Duoset kits (R&D Systems). For flow cytometry, skin was enzymatically digested to release cells and stained using a subset of antibodies. Cells were stained with Fixable Viability Dye eFluor780 (eBioscience), fixed in 4% methanol-free paraformaldehyde (Thermo Scientific) or Cytofix/Cytoperm (BD), and analyzed on a MACSQuant Analyzer 10 (Miltenyi). For cell sorting, cells were labeled with CD11b beads and sorted on magnetized columns (Miltenyi). For immunohistochemistry, skin was fixed before freezing in embedding medium and sectioned. Sections were blocked in Tris-Saline-Tween (TBS)/5% fish gelatin (Sigma-Aldrich) incubated with a primary antibody against Lyve-1 and the secondary chicken anti-goat IgG Alexa Fluor 647-conjugated antibody (Life Technologies).
5
Eosinophils Purification from Blood
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Eosinophils were purified from the peripheral blood from healthy donors using negative selection, as before (13 (link)). Briefly, venous blood was collected in a 6% dextran saline solution, and RBCs were allowed to sediment. Buffy coat was centrifuged over Ficoll-Paque (GE Healthcare, Pittsburgh, Pa) to separate granulocyte pellets. Eosinophils were isolated by means of incubation with a depletion antibody cocktail (StemSep; STEMCELL Technologies, Vancouver, British Columbia, Canada), followed by passage over magnetized columns (Miltenyi Biotec, Auburn, Calif). The purity of isolated eosinophils was greater than 98% of nucleated cells, and viability was greater than 99%.
6
Antigen-specific T cell enrichment
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Tetramer enrichment was used to isolate antigen-specific cells from pre-immune or acutely challenged mice. A modification of the method used by Obar et al. (Obar et al., 2008) (link) was employed. Following digestion with collagenase D, single-cell suspensions were prepared from the spleens (acutely challenged mice) or spleen and peripheral/mesenteric lymph nodes (pre-immune mice). When possible, the same tetramer (Trp2180-188/K b ) was used in both APC and PE to ensure specificity. Anti-PE and anti-APC beads and magnetized columns (both from Miltenyi Biotec) were used to enrich for tetramer-bound cells. Samples were stained and analyzed by flow cytometry; CountBright counting beads (Invitrogen) were used for enumeration.
7
Separation and Characterization of Malaria Parasites
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Mixed ring and trophozoite stage parasites were passed through a magnetized column (Miltenyi) to separate trophozoites from rings. Whole trophozoite infected erythrocytes were resuspended in about 30 pellet volumes of SDS sample buffer and fractionated on 4–12% gels (Life Technologies) by SDS PAGE. The ring samples were lysed in 0.09% saponin, and washed in PBS to remove haemoglobin. The ring pellet was similarly prepared for SDS-PAGE. After transfer to nitrocellulose membrane the blots were probed with rabbit serum for KAHRP and SBP1 (kind gifts from Alan Cowman and Brian Cooke) at 1/500 in 1% casein. Rabbit serum for Hsp70-1 was produced by the Walter & Eliza Hall Institute Monclonal facility and was similarly used at 1/1000. Mouse monoclonals for RESA 18.2 (a kind gift from Robin Anders) and EXP2 were used at 3 μg/mL. Secondary antibodies labeled with 700 and 800 nm fluorescent dyes were used at 1/5000 and detected with an Odyssey FC scanner (Li-Cor). Densitometry was performed using the Odyssey Image Studio software.
8
SARS-CoV-2 Tetramer Staining Protocol
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Tetramer staining was carried out as previously described (16 (link), 18 ). In brief, 30 to 90 million CD3 or CD4 enriched T cells were stained at room temperature for 1 hour using 5 ug of each tetramer in 50ul reaction. Tetramer tagged cells were enriched by adding anti-PE and/or anti-APC magnetic beads and passing the mixture through a magnetized column (Miltenyi). The tetramer-enriched samples were stained with live/dead dyes, exclusion markers (anti-CD19 and anti-CD11b, BioLegend), and other surface markers (Table S5 ) for 30 minutes at 4°C. Samples were acquired by flow cytometry using LSRII (BD) or sorted on FACS Aria (BD). Frequency calculation was obtained by mixing 1/10th of sample with 200,000 fluorescent beads (Spherotech) for normalization (16 (link)). Non-zero populations were included in the analyses performed using FlowJo (BD). Spectral flow cytometric analyses were performed with following modifications: 2ug of tetramers loaded with each of the twelve SARS-CoV2 peptides used in this study were tagged to the same fluorochrome and combined in the staining reaction. Tetramer enriched cells were stained with live/dead dyes, exclusion markers, and a panel of additional surface antibodies (Table S5 ) for 1h at 4°C followed by fixation with 2% paraformaldehyde. Samples were acquired on spectral flow Cytex AURORA (ARC 1207i).
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