Anti fitc magnetic microbeads

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Anti-FITC magnetic microbeads are small, magnetic particles coated with antibodies that bind specifically to the fluorescein isothiocyanate (FITC) molecule. These microbeads can be used to isolate or enrich FITC-labeled cells, proteins, or other biomolecules from complex samples, facilitating downstream analysis and applications.

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Magnetic microbeads (147 citations) Anti-FITC microbeads (100 citations) Anti-FITC conjugated magnetic MicroBeads (3 citations)

15 protocols using anti fitc magnetic microbeads

Separation and Characterization of IgA-Associated Gut Bacteria

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According to their association with IgA subclasses, pellets were processed to separate bacteria fractions, as previously reported (23 (link)). The pellets were incubated for fifteen minutes at room temperature with PBS containing 0.25% BSA, 5% FCS, and 2 mM EDTA. Subsequently, they were washed five times with PBS and resuspended in a final volume of 1 mL. Total sample volume was separated into four sterile tubes: a) total bacteria, b) IgA- bacteria, c) IgA1+ bacteria, and d) IgA2+ bacteria. For the separation of the IgA-associated bacterial fractions, the samples were incubated with biotinylated anti-human IgA1 (Cat# ab99796, Abcam^®) and anti-human IgA2 (Cat# ab128731, Abcam^®) at a dilution of 1:2500 and 1:2,000, respectively. Subsequently, streptavidin-APCy7 (Cat# 405208, Biolegend^®) or streptavidin-FITC (Cat# 405201, Biolegend^®) were added. Finally, magnetic anti-FITC microbeads (Cat# 130-048-701 Miltenyi Biotec Inc., Sunnyvale, CA, USA) or anti Cy7 microbeads (Cat# 130-091-652 Miltenyi Biotec Inc.) were added. Positive selection was carried out using a magnet system for cell separation (Cat# 18000 EasySep™ Magnet, Stem Cell Technologies^®, Canada). The purity of IgA1+, IgA2+, and IgA- bacterial fractions was verified in the CytoFLEX flow cytometer (Cat# B53000 Beckman Coulter^®) and analyzed with CytExpert Software (Beckman Coulter^®) (Supplementary Figure 2).

Sánchez-Salguero E., Corona-Cervantes K., Guzmán-Aquino H.A., de la Borbolla-Cruz M.F., Contreras-Vargas V., Piña-Escobedo A., García-Mena J, & Santos-Argumedo L. (2021). Maternal IgA2 Recognizes Similar Fractions of Colostrum and Fecal Neonatal Microbiota. Frontiers in Immunology, 12, 712130.

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Isolation of B cells and cDCs from Mouse Spleen

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B cells were purified from spleens using Ficoll® Paque Plus (GE Healthcare) gradient centrifugation and negative depletion with FITC-conjugated mAb specific for CD4 (GK1.5), Ly-76 (TER119) and CD43 (S7) and magnetic anti-FITC MicroBeads (Miltenyi Biotec). Preparations were approximately 95–98% pure for CD19⁺ B220⁺ B cells. Splenic cDCs were purified from mice subcutaneously injected with Flt3L-secreting melanoma cells (Mach et al., 2000 (link)), nine days before purification. cDCs were purified from spleens of Flt3L-expanded mice following spleen digestion with DNase I (Roche) and collagenase type III and Nycodenz® density gradient centrifugation with subsequent negative depletion using rat mAb specific for CD3 (KT3-1.1), Thy1 (T24/31.7), Ly-76 (Ter119), B220 (RA3-6B2) and Ly-6C/G (RB6-8C5) and anti-rat IgG-coupled magnetic beads (Qiagen) as previously described (Vremec, 2010 ). Preparations were approximately 90–95% pure for CD11c⁺ MHC II⁺ cDCs.

Schriek P., Liu H., Ching A.C., Huang P., Gupta N., Wilson K.R., Tsai M., Yan Y., Macri C.F., Dagley L.F., Infusini G., Webb A.I., McWilliam H.E., Ishido S., Mintern J.D, & Villadangos J.A. (2021). Physiological substrates and ontogeny-specific expression of the ubiquitin ligases MARCH1 and MARCH8. Current Research in Immunology, 2, 218-228.

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Isolation of ependymal cells from spinal cord

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Clec9a^CreRosa^LSLtdTomato mice were sacrificed with i.p. injection of pentobarbital, perfused with 20ml of sterile PBS, spinal cords dissected and digested into a single-cell suspension using Neural Tissue Dissociation kit (P) (Miltenyi Biotec) following manufacture instructions. Sample was enriched for ependymal cells by further processing with myelin removal magnetic beads II (Miltenyi Biotec) to remove myelinated cells by passage through a MACS column (Miltenyi Biotec). Cells were then stained with FITC-conjugated anti-CD45.2 (clone 104), incubated with anti-FITC magnetic MicroBeads (Miltenyi Biotec) and put through another MACS column to remove CD45⁺ cells. Cells were finally stained with APC-conjugated CD45 (clone 30-F11) and DAPI (to exclude dead cells) and single DNGR-1 traced (tdTomato⁺) CD45^- DAPI^- cells were FACS sorted on an Aria Fusion (BD) with a 100μm nozzle. QC confirmed viability <95% and cells were immediately loaded onto 10X Genomics Chromium according to manufacture instructions.

Frederico B., Martins I., Chapela D., Gasparrini F., Chakravarty P., Ackels T., Piot C., Almeida B., Carvalho J., Ciccarelli A., Peddie C.J., Rogers N., Briscoe J., Guillemot F., Schaefer A.T., Saúde L, & Reis e Sousa C. (2022). DNGR-1-tracing marks an ependymal cell subset with damage-responsive neural stem cell potential. Developmental Cell, 57(16), 1957-1975.e9.

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Isolation of Naive CD8+ T Cells

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This was performed as previously reported ^{9 (link), 10 (link)}. Briefly, inguinal,
axillary, brachial, cervical, and mesenteric lymph nodes (LNs) were harvested from WT OT-I
or IL-12RKO OT-I mice, pooled, and disrupted to obtain a single cell suspension. Cells
were incubated with FITC-labeled antibodies specific for CD4, B220, I-A^b, and
CD44. Anti-FITC magnetic MicroBeads (Miltenyi Biotech, Auburn CA) were then added and the
suspension passed through separation columns attached to a MACS magnet. Cells that did not
bind were collected with a purity >95% CD8⁺ cells and
<0.5% CD44^hi cells.

Garcia K., Sun Z., Mattson E., Li L., Smyth K, & Xiao Z. (2014). IL-12 is required for mTOR regulation of memory CTLs during viral infection. Genes and immunity, 15(6), 413-423.

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Magnetic Sorting of GEP-expressing Cells

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Isolation of GEP-expressing cells was performed by magnetic activated cell sorting (Miltenyi Biotec) according to manufacturer's instructions. Briefly, cells were stained with FITC-conjugated mouse anti-human GEP (Versitech Ltd., [14 (link)]), and then incubated with anti-FITC magnetic microbeads (Miltenyi Biotec), prior to magnetic separation. Cells were sorted based on the surface expression of GEP, but not on their intracellular expression, because permeabilization was avoided to keep the cells viable for subsequent functional assays. After cell isolation, sorted cells were assessed for cell viability by trypan blue staining and total cellular GEP levels by flow cytometry using mouse anti-human GEP (R&D systems, Minneapolis, MN) antibody recognizing different GEP epitope. Sorted cells were permeabilised prior to staining so that total GEP expression of the sorted cells could be determined. Cells were then stained with PE-conjugated rabbit anti-mouse secondary antibody. Post-sorting analysis consistently indicated purities >80% with minimal cell death.

Cheung P.F., Cheung T.T., Yip C.W., Ng L.W., Fung S.W., Lo C.M., Fan S.T, & Cheung S.T. (2016). Hepatic cancer stem cell marker granulin-epithelin precursor and β-catenin expression associate with recurrence in hepatocellular carcinoma. Oncotarget, 7(16), 21644-21657.

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Antigen Presentation Assay with CFSE-labeled T Cells

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OT-1 and OT-2 mice were sacrificed and single cell suspensions were obtained from spleen and LNs. OT-1 CD8⁺ and OT-2 CD4⁺ cells (≥87% pure) were obtained after incubation with anti-CD8β.2 fluorescein isothiocyanate (FITC) monoclonal antibody (mAb) (BD) and CD4 FITC mAb (BD), respectively, followed by positive selection with anti-FITC magnetic microbeads (Miltenyi Biotec). Cells were labeled for 8 min at room temperature with 2.5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Eugene, OR, USA). For antigen presentation assays, BMdDCs were kept for 2 days in complete Opti-MEM medium with GM-CSF at 20 ng/ml, then incubated for 5 h with Ovalbumin (OVA, Hyglos GmbH, Resenburg, Germany) at 0.2 mg/ml, in the presence or not of SCF at 100 ng/ml. After extensive washings, BMdDCs (200–250 × 10³ cells/well) were cocultured in flat-bottom 96-well plates in complete Opti-MEM medium with 5% FCS with either purified CFSE-labeled OT-1 CD8⁺ (700–750 × 10³ cells/well) or purified CFSE-labeled OT-2 CD4⁺ (200 × 10³ cells/well) cells for 3 days. CFSE dilution by TCR⁺ CD8⁺ and TCR⁺ CD4⁺ cells was evaluated by flow cytometry (43 (link)–45 (link)).

Barroeta Seijas A.B., Simonetti S., Vitale S., Runci D., Quinci A.C., Soriani A., Criscuoli M., Filippi I., Naldini A., Sacchetti F.M., Tarantino U., Oliva F., Piccirilli E., Santoni A, & Di Rosa F. (2017). GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells. Frontiers in Immunology, 8, 147.

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Sorting and Isolating ASCs and FAP from SVF

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ASC sorting: ScAT- or PGAT- derived SVF were depleted in CD45⁺ and CD31⁺ cells using anti-CD45-FITC (Miltenyi, 130-116-535) and anti-CD31-FITC antibodies (Miltenyi, 130-123-675) followed by anti-FITC magnetic microbeads (Miltenyi, 130-048-701) using an autoMACS® Pro Separator (MACS Cell Separation, Miltenyi Biotec SAS) according to the manufacturer’s instructions. CD45⁻/CD31⁻ cells were then positively sorted for Sca-1 with anti-Sca-1 magnetic microbeads (Miltenyi, 130-106-641). Isolated ASCs were used for RNAseq experiments, for muscle injection in platelet-depleted or lipectomied animals (see methods), or for in vitro platelet/ASCs interaction challenges (see methods). FAP sorting: muscle-derived SVF were treated as above and used for RNAseq experiments, affymetrix analysis (ctrl or injured), and DNA extraction.

Sastourné-Arrey Q., Mathieu M., Contreras X., Monferran S., Bourlier V., Gil-Ortega M., Murphy E., Laurens C., Varin A., Guissard C., Barreau C., André M., Juin N., Marquès M., Chaput B., Moro C., O’Gorman D., Casteilla L., Girousse A, & Sengenès C. (2023). Adipose tissue is a source of regenerative cells that augment the repair of skeletal muscle after injury. Nature Communications, 14, 80.

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Isolation and Sorting of Dendritic Cell Subsets

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For ex vivo DC isolation, the thymuses or spleens were pooled and cut with blunt scissors before digestion in Roswell Park Memorial Institute (RPMI) 1640 with 2% fetal bovine serum (FCS) supplemented with Liberase (200 μg/ml, Roche) and DNase I (40 μg/ml, Sigma-Aldrich) for 10–15 min. After incubation with 2.4G2 antibody, DCs were isolated using CD11c MicroBeads (Miltenyi Biotec), according to manufacturer's instruction. Each of the cDC subsets and pDCs was subsequently examined by fluorescence-activated cell sorting using a FACSAria™ II high-speed cell sorter (BD Biosciences) following staining with F4-80-, CD45.1- or CD45.2-, CD11c-, CD11b-, B220-, CD24-, and Sirpα-specific antibodies.
For pre-cDC and pDC isolation, BM was extracted from the femurs and tibias, and erythrocytes removed using Gey's treatment. Lineage cells were first removed using fluorescein isothiocyanate (FITC) conjugate anti-CD3, anti-CD19, anti-NKP46, and, for pre-cDC isolation only, anti-B220, and anti-FITC magnetic MicroBeads (Miltenyi Biotec). Pre-cDCs and pDCs were subsequently FACS-sorted following staining with anti-MHC II-, CD135-, Sirpα- and CD11c-specific antibodies or CD19-, B220-, and CD11c-specific antibodies, respectively.

Mahiddine K., Hassel C., Murat C., Girard M, & Guerder S. (2020). Tissue-Specific Factors Differentially Regulate the Expression of Antigen-Processing Enzymes During Dendritic Cell Ontogeny. Frontiers in Immunology, 11, 453.

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Purification and Activation of γδ T Cells

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To purify γδ T cells, CD4⁺ T cells were first depleted from single cell suspensions of the LNCs derived from naïve WT and CD69KO mice by using anti-mouse CD4 magnetic microbeads (Miltenyi Biotec, Gladbach, Germany) in combination with MACS columns (Miltenyi Biotec). It has been demonstrated that activated γδ T cells amplify Th17 response [14] (link). Hence, this CD4⁺ cell-depletion step can minimize the participation of Th17-derived IL-17 in the assay. Then, the CD4⁻ LNCs were incubated with FITC-conjugated anti-mouse TCRγδ antibody (eBioscience, Santa Clara, CA) followed by a reaction with anti-FITC magnetic microbeads (Miltenyi Biotec). The cells selected by MACS columns were used as γδ T cells and cultured in RPMI 1640 medium (Sigma) supplemented with 10% FBS, 500 µM 2-mercaptoethanol, penicillin (100 units/ml), and streptomycin (100 µg/ml) for 24 h at 37 °C in a humidified atmosphere (5% CO₂). Then, according to a previous report [14] (link), γδ T cells were stimulated with IL-1β (10 ng/ml) and IL-23 (10 ng/ml) for 72 h. The supernatants were subjected to enzyme-linked immunosorbent assay (ELISA) for mouse IL-17A (Biolegend). We confirmed that IL-17A levels in the supernatants from unstimulated γδ T cells of the two genotypes were below the detection limit.

Fujita T., Yoshioka K., Umezawa H., Tanaka K., Naito Y., Nakayama T., Hatano M., Tatsumi K, & Kasuya Y. (2016). Role of CD69 in the pathogenesis of elastase-induced pulmonary inflammation and emphysema. Biochemistry and Biophysics Reports, 7, 400-407.

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Isolation of Naive CD8+ T Cells

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This was performed as in previous reports^{22 (link)}; briefly, inguinal, axillary, brachial, cervical, and mesenteric lymph nodes (LNs) were harvested from WT OT-I or B6 mice, pooled, and homogenized to obtain single cell suspensions. CD8⁺CD44^lo cells were enriched by negative selection using MACS magnetic beads (Milteny Biotec, Auburn, CA) wherein cells were coated with FITC-labeled antibodies specific for CD4, B220, I-A^b, and CD44 (Biolegend San Diego, CA). Anti-FITC magnetic MicroBeads (Miltenyi Biotech) were added, and the suspension passed through separation columns attached to a MACS magnet. Cells that did not bind were collected, and were >95% CD8⁺ and <0.5% CD44^hi.

Li L., Jay S.M., Wang Y., Wu S.W, & Xiao Z. (2017). IL-12 stimulates CTLs to secrete exosomes capable of activating bystander CD8+ T cells. Scientific Reports, 7, 13365.

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