Anti fitc microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany, Canada, United States

Anti-FITC microbeads are magnetic particles coated with anti-FITC (Fluorescein Isothiocyanate) antibodies. They are designed for the isolation and enrichment of FITC-labeled cells, proteins, or other biomolecules from complex samples.

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Anti-FITC magnetic microbeads (15 citations) Anti-F4/80-FITC/anti-FITC-microbeads (3 citations) MACS™ anti-FITC microbeads (3 citations) Anti-FITC conjugated magnetic MicroBeads (3 citations) Anti-FITC or PE ferromagnetic microbeads (2 citations) Anti‐FITC MultiSort microbeads (2 citations) Anti-Alexa Fluor 647 or anti-FITC microbeads (2 citations)

100 protocols using anti fitc microbeads

Immune Cell Isolation and Purification

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Macrophages were purified by using an anti-F4/80 FITC-conjugated antibody and anti-FITC microbeads (Miltenyi Biotec) according to the manufacturer’s protocol. CD8⁺ T cells were purified by using an anti-CD8 APC-conjugated antibody and anti-APC microbeads (Miltenyi Biotec). PD1⁺ cells were purified by using an anti-PD-1 FITC-conjugated antibody and anti-FITC microbeads (Miltenyi Biotec).

Kado T., Nawaz A., Takikawa A., Usui I, & Tobe K. (2019). Linkage of CD8+ T cell exhaustion with high-fat diet-induced tumourigenesis. Scientific Reports, 9, 12284.

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Isolation and Transfer of VRC01 B Cells

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Lymphocytes were prepared from spleens of VRC01^gHL by density gradient centrifugation (Histopaq, Sigma). VRC01^gHL B cells were phenotyped for eOD-GT8 probe binding prior to transfer in each experiment. B cells were purified by negative selection by magnetic depletion using FITC conjugated anti-CD43 (clone S7), anti-FITC microbeads (Miltenyi Biotec), and an LS column (Miltenyi Biotec). Cells were enumerated on a hemocytometer and transferred retro-orbitally (RO) into recipient hosts. All steps were performed in transfer buffer (5% Horse Serum/Dulbecco’s PBS (with calcium and magnesium)). All tubes, pipette tips, and syringe needles were pre-coated for at least an hour at room temperature in transfer buffer to minimize cell loss. Mice were rested for approximately 24 hours before immunization. Cells used for transfers were regularly checked for health by using an aliquot for various in vitro stimulation assasy. For CFSE labeling, purified B cells were reacted with 10μM CFSE for 8 minutes with gentle rocking at room temperature and then quenched with two washes of transfer buffer and enumerated after labeling.

Abbott R.K., Lee J.H., Menis S., Skog P., Rossi M., Ota T., Kulp D.W., Bhullar D., Kalyuzhniy O., Havenar-Daughton C., Schief W.R., Nemazee D, & Crotty S. (2017). Precursor Frequency and Affinity Determine B Cell Competitive Fitness in Germinal Centers, Tested with Germline-Targeting HIV Vaccine Immunogens. Immunity, 48(1), 133-146.e6.

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Isolation and Purification of CXCR5+ CD4+ T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation with Ficoll-Hypaque solution (Haoyang Biological Technology Co, Tianjin, China). CD4⁺ T cells were isolated from PBMCs through immunomagnetic cell sorting using negative selection kits, and CXCR5⁺ cells were purified from CD4⁺ T cells by FITC-conjugated anti-human CXCR5 mAb and anti-FITC microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.

Yang J., Geng L., Ma Y., Tang X., Peng H., Tian J., Xu H, & Wang S. (2021). SLAMs Negatively Regulate IL-21 Production in Tfh-Like Cells from Allergic Rhinitis Patients. Journal of Asthma and Allergy, 14, 361-369.

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Enrichment and Transfer of MAIT Cells

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Thymic cells from B6-MAIT^cast mice were stained with an anti–CD24-FITC antibody and depleted of the CD24⁺ (HSA⁺) fraction using anti-FITC microbeads (Miltenyi) and LS columns (Miltenyi) according to the manufacturer’s instructions. Three thymuses were pooled for injection into one recipient, and the cell concentration was adjusted to transfer ∼40,000 MAIT cells among total enriched thymic cells. Cells were transferred intravenously into B6-MAIT^cast recipients, and the spleen, the liver, and the lungs were collected 36 h later for flow cytometry analysis.

Salou M., Legoux F., Gilet J., Darbois A., du Halgouet A., Alonso R., Richer W., Goubet A.G., Daviaud C., Menger L., Procopio E., Premel V, & Lantz O. (2019). A common transcriptomic program acquired in the thymus defines tissue residency of MAIT and NKT subsets. The Journal of Experimental Medicine, 216(1), 133-151.

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Magnetic Sorting of hPDLSCs

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The hPDLSCs were sorted using magnetic-activated cell sorting (MACS) as per the manufacturer's protocol (Miltenyi Biotec, Gladbach, Germany). Briefly, the hPDL cell suspension (5 × 10 6 ) was labeled with a fluorescein isothiocyanate (FITC)-conjugated antibody (anti-human SSEA-4, 5 μL/10 6 cells, clone MC-813-70, FITC; STEMCELL Technologies, Vancouver, BC, Canada) and anti-FITC Microbeads (5 μL/10 6 cells; Miltenyi Biotec) were used to detect the FITC on the primary antibody. The cell suspension was applied onto the prepared magnetic sorting (MS) column (15) . The cells were then sorted into labeled and unlabeled fractions using a MACS column (Miltenyi Biotec).

Yoo J.H., Lee S.M., Bae M.K., Lee D.J., Ko C.C., Kim Y.I, & Kim H.J. (2018). Effect of orthodontic forces on the osteogenic differentiation of human periodontal ligament stem cells. Journal of oral science, 60(3).

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Enrichment of Leukemic Stem Cells

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Human AML cell line KG1a (ATCC, Manassas, VA) was maintained in suspension culture with Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL of penicillin and streptomycin. Since LSCs were defined as CD34+CD38− fraction of KG1a cells and all of KG1a cells were CD34+, LSCs were enriched by indirectly labeling with CD38-FITC antibody and anti-FITC microbeads according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, cell suspensions were centrifuged at 125 g for 10 min and washed in phosphate-buffered saline (PBS). Then, cell pellets were resuspended in separating buffer (PBS containing 0.5% bovine serum albumin) and incubated with CD38-FITC antibody for 30 min. After washing with PBS, cell pellets were resuspended in separating buffer containing anti-FITC microbeads for 15 min. After several washing steps, filtrates (CD34+CD38− cells) were collected by a LD column using a MidiMACS separator system. Throughout the sorting procedure, cells were kept at 4°C and analyzed immediately by flow cytometry. The remaining cells after depletion of LSCs were defined as AML cells, which were used for coculture studies.

Wang Y., Cheng Q., Liu J, & Dong M. (2016). Leukemia Stem Cell-Released Microvesicles Promote the Survival and Migration of Myeloid Leukemia Cells and These Effects Can Be Inhibited by MicroRNA34a Overexpression. Stem Cells International, 2016, 9313425.

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Purification and Activation of αβ and γδ T Cells

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αβ T cells were purified from B6 mice immunized with the human interphotoreceptor retinoid-binding protein (IRBP) peptide IRBP_1-20, as described previously [4 (link),7 (link),8 (link)], while γδ T cells were purified from immunized and control (naïve) B6 or A2AR^-/- mice. Nylon wool-enriched splenic T cells from naïve or immunized mice were incubated sequentially for 10 min at 4°C with FITC-conjugated anti-mouse γδ TCR or αβ TCR Abs and 15 min at 4°C with anti-FITC Microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany); the cells were then separated into bound and non-bound fractions on an autoMACS^TM separator column (Miltenyi Biotec GmbH). The purity of the isolated cells was >95%, as determined by flow cytometric analysis using PE-conjugated Abs against αβ or γδ T cells. Resting γδ T cells were prepared either by isolation from naïve mice or by incubating activated γδ T cells in cytokine-free medium for 5–7 d, at which time they show down-regulation of CD69 expression [3 (link)]. Highly activated γδ T cells were prepared by incubating resting γδ T cells for 2 d with Abs against the γδ TCR (GL3) and CD28 (both 2 μg/ml, both from Bio-Legend, San Diego, CA), or cytokine combination (IL-1,IL-7 and IL-23).

Liang D., Shao H., Born W.K., O'Brien R.L., Kaplan H.J, & Sun D. (2018). High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU. PLoS ONE, 13(6), e0199601.

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Isolation of CD4+ and CD8+ T Cells

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Peripheral blood mononuclear cells (PBMCs) from healthy donors were purified from buffy coats (Scottish Blood Transfusion Service) by density gradient centrifugation following manufacturer’s instructions (Lymphoprep, StemCell Technologies, #07801). For CD4⁺ and CD8⁺ T cells isolation, 1 × 10⁸ PBMCs per donor were stained with anti-CD4^FiTC (Biolegend, #357406) or anti-CD8^FITC (Biolegend, #344704) antibodies and isolated by magnetic activated cell sorting (MACS, Miltenyi) using anti-FiTC microbeads (Miltenyi, #130-048-701) according to manufacturer’s instructions to a purity ≥ 99%.

Martinez-Fabregas J., Wang L., Pohler E., Cozzani A., Wilmes S., Kazemian M., Mitra S, & Moraga I. (2020). CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci. Cell Reports, 33(12), 108545.

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Purification and Differentiation of T Cell Subsets

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CD4⁺ T cells were purified from spleens by magnetic‐activated cell sorting (Dynal CD4⁺ T cell negative isolation kit, Invitrogen, Gent, Belgium) according to the manufacturer's protocol. Naive or memory T cells were purified from previously isolated CD4⁺ T cells subsets by positive or negative selection of CD62L‐expressing cells using the magnetic cell sorting kit (CD62L microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. For Treg differentiation experiments, CD25‐positive cells were removed from purified CD4⁺ T cells by magnetic‐activated cell sorting using FITC‐conjugated anti‐CD25 antibodies and anti‐FITC microbeads (Miltenyi Biotec).

Weatherly K., Bettonville M., Torres D., Kohler A., Goriely S, & Braun M.Y. (2015). Functional profile of S100A4‐deficient T cells. Immunity, Inflammation and Disease, 3(4), 431-444.

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Enrichment of Ductal Cells via DBA Lectin Sorting

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To enrich extracted cell populations for ductal cell extracts, DBA Lectin sorting was completed. Cells were isolated from a mouse pancreata as described above and suspended with 1:200 diluted DBA lectin–FITC (Vector Labs, cat. no. FL-1031) and incubated for 10 min in the dark at 4 °C on a rotor. Cells were washed with 700 μl of separation buffer (PBS, 0.5% BSA, and 2mM EDTA) and centrifuged for 10 min at 300g at 4 °C. The pellet was resuspended in 90 μl of separation buffer and 10 μl of anti-FITC MicroBeads (Miltenyi Biotec, cat. no. 130-048-701) and incubated on a rotor at 4 °C for 15 min. The cells were washed with 1mL of sorting buffer (PBS 0.5% BSA, 2mM EDTA) and the pellet (300g for 10 min) was resuspended in 500 μl of sorting buffer. MACS Separation (MS) columns (Miltenyi Biotec, cat. no. 130-042-201) were prepared by running 500 μl of chilled sorting buffer and then the labeled cell suspensions (500 μl) were applied to the column. The columns were washed three times with 500 μl of buffer and the flow through was collected as the DBA lectin–negative fraction. The column was removed from the magnetic field and 1,000 μl of separation buffer was then added with a plunger to collect the DBA lectin–positive fraction. The samples were spun down (300g for 15 min at 4 °C) and the pellets used for downstream applications.

Das K.K., Heeg S., Pitarresi J.R., Reichert M., Bakir B., Takano S., Kopp J.L., Wahl-Feuerstein A., Hicks P., Sander M, & Rustgi A.K. (2018). ETV5 regulates ductal morphogenesis with Sox9 and is critical for regeneration from pancreatitis. Developmental dynamics : an official publication of the American Association of Anatomists, 247(6), 854-866.

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