For gene-expression analysis by qRT-PCR, CD49b+ splenic NK cells were enriched by magnetic sorting (Miltenyi Biotec), were purified by flow cytometry (BD FACSAria II) (NK1.1+CD49b+CD3−CD19−) and were frozen in RLT buffer (Qiagen). RNA was extracted with an RNeasy Mini kit (Qiagen). cDNA was synthesized from RNA with Superscript III first-strand synthesis system for RT-PCR (Invitrogen). cDNA expression was analyzed by quantitative PCR using iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories) and a StepOnePlus system (Applied Biosystems). The expression of target genes was calculated and normalized to the expression of the control gene Gapdh using the 2−ΔΔCT method.
Magnetic sorting
Manufactured by Miltenyi Biotec
Sourced in Germany
Magnetic sorting is a laboratory technique that utilizes magnetic particles to isolate and separate specific target cells or molecules from a complex sample. The core function of this process is to enable the efficient and accurate selection and extraction of desired components from a heterogeneous mixture.
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Lab products found in correlation
Collagenase b, by Roche (4 mentions)
Pe conjugated anti ly6c antibody hk1, by Thermo Fisher Scientific (4 mentions)
Apc conjugated f4 80 antibody bm8, by Thermo Fisher Scientific (4 mentions)
Fitc conjugated ly6g antibody 1a8, by BioLegend (4 mentions)
Collagenase 2, by Thermo Fisher Scientific (3 mentions)
Facsaria 3 sorter, by BD (3 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Rat fibronectin, by Merck Group (2 mentions)
Vitronectin, by Merck Group (2 mentions)
Cytation 3, by Agilent Technologies (2 mentions)
Grgdnp peptide, by Enzo Life Sciences (2 mentions)
Facsaria 2, by BD (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Elisa plate, by Greiner (1 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)
Anti nk1.1 pk136, by BioLegend (1 mentions)
25 protocols using magnetic sorting
1
Gene Expression Analysis of NK Cells
2
Recipient CIITA^Tg PIV^−/− Mice Bone Marrow Transplant
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Recipient CIITATg PIV−/− mice were exposed to total body irradiation of 900 rad from a [137Cs] source in two split doses that were given 4 h apart. The mice were then rested for 24 h before being administered bone marrow cells. Total bone marrow cells were extracted from the femurs and tibiae of donor mice, and T cells were depleted by the magnetic sorting method (Miltenyi Biotech, USA). Each recipient mouse received 3 × 106 T-cell-depleted bone marrow cells in a volume of 300 ml of PBS via lateral tail vein injection, and the thymus and spleen were analyzed 8 weeks later.
3
Muscle Injury and Immune Cell Isolation
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WT or Nur77 KO mice were anesthetized with 2.5% isoflurane using a SomnoSuite device. After anesthesia, muscle damage was induced in the tibialis anterior (TA) muscle by injecting 50 µL of 12 µM cardiotoxin (Latoxan, Valence, France) in phosphate-buffered saline (PBS). Mice were sacrificed and muscles were collected on days 2, 3, and 4 post-injury and processed for cell and mRNA analysis. TA muscles were dissociated in RPMI containing 0.2% collagenase II (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 1 h and filtered through a 100 µm and then a 40 µm filter. Muscle-derived CD45+ cell isolation was carried out as described earlier (50 (link)). CD45+ cells were separated using magnetic sorting (Miltenyi Biotec, Gladbach, Germany).
4
NK Cell Stimulation and Functional Assays
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CD49b+ NK cells were enriched by magnetic sorting (Miltenyi) from BM. For stimulation through NK1.1, 10 µg/ml anti-NK1.1 (PK136, 108701; BioLegend) in NaHCO3 (pH 9.2) was precoated on an ELISA plate (655081; Greiner Bio-One). After culture in 20 ng/ml murine IL-15 (210-15; PeproTech) overnight, NK cells were then added to the plate with Brefeldin A (555029; BD Biosciences) for the last 4.5 h. For stimulation through cytokines, NK cells were cultured with 1,000 U/ml IL-2 (212-12; PeproTech) and 10 ng/ml IL-12 (210-12, PeproTech) overnight and Brefeldin A for the last 4.5 h. Cytofix/Cytoperm Fixation/Permeabilization Kit (554714; BD Biosciences) was used to detect IFN-γ (XMG1.2, 12-7311-81; eBioscience), and Cyto-Fast Fix/Perm Buffer Set (426803; BioLegend) was used to detect GZMB (QA16A02, 372207; BioLegend) according to the manufacturer’s instructions.
5
Antigen-Specific CD4+ T-cell Isolation
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SJL/J mice (6–8 weeks old, female) were immunized subcutaneously with 100 μg of S18 emulsified in CFA. At the end of 2 weeks splenocytes were harvested and co-cultured with stimulators at a ratio of 1:1. Stimulators were prepared by loading healthy mice derived irradiated splenocytes with S18 (10 μg/ml). Co-cultures were plated (5 million cells/ml) in primary culture medium (DMEM supplemented with 10% FBS, 2 mM GlutaMax, Anti-anti, 0.5 μM β-mercaptoethanol and 10 U/ml of recombinant human IL-2 (Sigma)). Cultures were replenished with IL-2 every 3–4 days and re-stimulated weekly using stimulators. At the end of 2 weeks S18 reactive CD4+ T-cells were isolated from mixed culture by magnetic sorting (Miltenyi Biotec) and infused (20 million cells/mice) a day prior to EAE induction.
6
Isolation and Characterization of Muscle-Resident Immune Cells
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TA muscles from CTX-injured animals were carefully isolated with fine scissors and fascia was removed29 (link). Despite alterations in the integrity of the muscle tissue during injury (e.g., swelling), the tissue remains intact during collection. Muscles were weighed and then placed directly into ice-cold PBS. Muscles were then dissociated in RPMI containing 0.2% collagenase B (Roche Diagnostics GmbH) at 37°C for 1 hour and filtered through a 100 μm and a 40 μm filter. CD45+ cells were isolated using magnetic sorting (Miltenyi Biotec). For FACS, macrophages were incubated with Fcγ receptor blocking antibodies and with 10% normal rat serum: normal mouse serum 1:1 mix, then stained with a combination of PE-conjugated anti-Ly6C antibody (HK1.4, eBioscience), APC-conjugated F4/80 antibody (BM8, eBioscience) and FITC-conjugated Ly6G antibody (1A8, Biolegend). Ly6Chi F4/80lo Ly6Gneg macrophages, Ly6Clo F4/80hi Ly6Gneg macrophages and Ly6Ghi Ly6Cint F4/80neg neutrophils were quantified. In each experiment, samples were processed in parallel to minimize experimental variation. Cells were analyzed on a BD FACSAria III or MoFlo Astrios sorter (Summit v6 software) and data analysis was performed using BD FACSDIVA and FlowJo V10 software. Gating strategy is shown in Supplementary Fig. 2a –b .
7
Th17 Cell Migration Assay
8
Th17 Cell Migration Assay
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CD4+ T cells were isolated from spleen of C57Bl/6 or Itgb3−/− mice by negative selection and magnetic sorting (Miltenyi Biotech). To generate Th17 cells, T cells were cultured in RPMI 1640 medium supplemented with 10% FCS, IL-6, IL-23, IL-1β, and anti-IFN-γ antibody, with or without TGF-β1, in plates coated with anti-CD3 antibody. Cells were harvested between days 5–7 of culture and resuspended in migration medium (RPMI with 2% BSA). Migration was assayed in 96-well cell-permeable chambers (5-μm pore size polycarbonate membranes) which had been pre-coated by incubation with rat fibronectin (Sigma; 10 μg/ml), vitronectin (Sigma; 1 μg/ml) or no matrix at 37°C for 2 hr, and remaining protein binding sites blocked with RPMI/BSA for 1 hr. 4 × 104 T cells were added to the upper chambers, and RPMI containing 1% FCS was added to the lower chambers. In some cases, GRGDNP peptide (Enzo Life Sciences) was added to upper chambers at 2 μg/ml. After 2 hr, the upper chambers were removed, and migrated cells in the lower chamber were stained with Calcein AM (1 μM; Invitrogen) and visualized and counted by imaging plate reader (Cytation 3; BioTek).
9
NK Cell Isolation and RNA-seq
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NK cells were enriched from BM using biotin-CD49b antibody (DX5, 13-5971-85; eBioscience) by magnetic sorting (Miltenyi) followed by flow cytometric sorting for NK1.1+CD49b+ cells. Purified NK cells 2 × 104 were sorted into RNeasy lysis buffer and total RNA was isolated using the RNeasy Micro Kit (QIAGEN) according to the manufacturer’s directions. Libraries were constructed using Nugen’s Ultralow Library Systems and were subsequently subjected to 76 cycles of NextSeq 500 sequencing. Sequencing data were processed as described previously (Jacobsen et al., 2020 (link)). Data are available through the Gene Expression Omnibus (accession no. GSE156046 ).
10
Dexamethasone-Induced Phagocytosis by CD45+ Cells
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TA muscle was damaged as described above. Two and a half days after injury, mice were treated with i.p. injection of Dex (Sigma‐Aldrich, D2915, 1 mg/kg). At day 3, TA muscles were harvested, digested for 1 h with collagenase (Roche, 11088831001, 1 mg/ml) in RPMI medium at 37°C. Digested muscles were filtered through 30 μm filter with DMEM containing 50% FBS. CD45pos cells were isolated using magnetic sorting (Milteny Biotec, 130‐042‐201 and 130‐052‐301) and were seeded at 190,000 cells per wells in 12‐well plates in DMEM serum free. Primary myoblasts previously stained with PKH67 (Sigma‐Aldrich, MINI67) were first submitted to 20 mM of H2O2 for 20 min to induce necrosis before being added to wells at 1:3 ratio (570,000 myoblasts per condition). After 6 h of phagocytosis, cells were harvested, blocked with FcR Blocking Reagent (Miltenyi Biotec, 130‐059‐901) and thereafter stained with anti‐CD45 antibody (eBioscience, 25‐0451‐82, 1:200), anti‐Ly6C antibody (eBioscience, 12‐5932‐82, 1:400) and anti‐CD64 antibody (BD Pharmingen, 558539, 1:100) for flow cytometry analysis.
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