Imag cell separation magnet

Manufactured by BD

Sourced in United States

The BD IMag™ Cell Separation Magnet is a laboratory instrument designed for the separation and isolation of magnetically labeled cells. It utilizes a strong magnetic field to attract and retain magnetic particles, facilitating the separation of target cells from the surrounding sample.

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12 protocols using imag cell separation magnet

Isolation of Memory B Cells

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Starting from single-cell suspensions of splenocytes, memory-phenotype B cells (M_phenBC) were enriched by depleting IgD⁺ and Thy1.2⁺ cells using biotinylated antibodies followed by BD IMag™ streptavidin particles on an IMag™ Cell Separation Magnet (BD Biosciences, San Jose, CA). Cells were stained as described for flow cytometry and dump negative, IgD⁻ CD38⁺, B220⁺ cells were sorted into 10% FBS in PBS for downstream applications.

Brookens S.K., Cho S.H., Basso P.J, & Boothby M.R. (2020). AMPKα1 in B cells dampens primary antibody responses yet promotes mitochondrial homeostasis and persistence of B cell memory. Journal of immunology (Baltimore, Md. : 1950), 205(11), 3011-3022.

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Isolation and Co-culture of Murine T and B Cells

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Splenic CD4⁺ CD25⁻ T cells and B220⁺ B cells were isolated from BALB/c mice. Splenic B220⁺ B cells were immunomagnetically purified using a BD IMag Cell Separation Magnet. Splenic CD4⁺ CD25⁻ T cells were purified by negative immunomagnetic selection using the EasySep Mouse CD4⁺ T Cell Isolation Kit (STEMCELL Technologies). CD4⁺CD25⁺ tTreg cells were obtained by incubating PE‐anti‐CD25 (BioLegend) and anti‐PE beads (BD Biolegend). Purified B cells were co‐cultured with purified splenic CD4⁺ CD25⁻ T cells under anti‐CD3/CD28 (0.5 μg/ml) stimulation for 3 days. Treg‐of‐B cells were obtained by depleting B220⁺ B cells.

Huang J., Lin Y., Wang L, & Chiang B. (2023). M2‐like macrophages polarized by Foxp3− Treg‐of‐B cells ameliorate imiquimod‐induced psoriasis. Journal of Cellular and Molecular Medicine, 27(11), 1477-1492.

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Phagosome Isolation and Protein Analysis

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BMDMs were treated with magnetic beads (1μm diameter, Thermo Fisher Scientific, USA) and allowed to phagocytose the beads at 37 °C for 4 h. Cells were then washed twice with PBS, harvested in homogenization buffer (250 μM sucrose, 3 mM imidazole, pH 7.4), and homogenized by passage through 21G syringe for 25 strokes. Phagosomes containing the magnetic beads were isolated using IMag Cell Separation Magnet (BD Biosciences, USA) and washed twice with homogenization buffer. Phagosomes were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and protease inhibitors at 4 °C for 15 min. Lysates were spun down to clear debris, and protein quantification was performed with a BCA protein assay kit (Thermo Fisher Scientific, USA) following manufacturer’s instruction.

Song H.S., Park S., Huh J.W., Lee Y.R., Jung D.J., Yang C., Kim S.H., Kim H.M, & Kim Y.M. (2022). N-glycosylation of UNC93B1 at a Specific Asparagine Residue Is Required for TLR9 Signaling. Frontiers in Immunology, 13, 875083.

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Inducing Plasma Cell Differentiation in Splenic B Cells

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Splenic B cells were purified from Rosa26-CreER^T2 mice (Prkaa1 +/+ and Prkaa1 f/f) by negative selection using biotinylated anti-CD43, -Thy1.2, and -F4/80 (>85% CD19⁺) followed by streptavidin particles and IMag™ Cell Separation Magnet (BD Biosciences). To induce plasma cell differentiation, B cells were seeded at 5 × 10⁵ per mL and treated with 5 μg/mL LPS (Sigma), 10 ng/mL BAFF (AdipoGen, San Diego CA), 10 ng/mL IL-4 (Peprotech, Rocky Hill, NJ), 5 ng/mL IL-5 (Peprotech), and 50 nM 4-hydroxy-tamoxifen (4-OHT) (Sigma). Cells were cultured for 2–8 days in RPMI-1640 supplemented with 10% FBS (Peak, Denver, CO), 100 U/mL penicillin (Invitrogen), 100 μg/mL streptomycin (Invitrogen), 3 mM L-glutamine (Invitrogen), and 0.1 mM 2-mercaptoethanol (Sigma). B cells were also expanded on the NB21 feeder line as previously described (, 30 ). Every three days, supernatants were frozen for further analysis and the expanded B cells were reseeded on fresh NB21 feeder cells in new media and 4-OHT. For spontaneous antibody secretion, day 8 LPS cultures or day 9 NB21.2D9 cultures were subjected to Ficoll spin (Invitrogen) to eliminate dead cells and debris. Cells were then washed and 5 × 10⁴ cells were seeded in 100 μL of fresh media in a 96 well plate for 8 hours. Supernatants were frozen and levels of secreted IgG1 were determined by ELISA.

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Evaluating SF's Effect on CD4+ T Cells

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CD4⁺ T cells were isolated from the splenocytes of 6-to-8-week-old female C57BL/6 mice using anti-mouse CD4 magnetic particles and an IMag Cell Separation Magnet (BD Biosciences, Franklin Lakes, NJ, USA). To evaluate the effect of SF on the viability of CD4⁺ T cells, the cells were treated with different concentrations of SF (0.1–1 mg/mL) for 72 h, and cell viability was detected with a cell counting kit-8.

Hua D., Yang J., Meng Q., Ling Y., Wei Q., Wang Z., Wei Q., Chen J., Ye J., Han X., Su K., Kong W., Xu C., Cao P, & Hu C. (2021). Soufeng sanjie formula alleviates collagen-induced arthritis in mice by inhibiting Th17 cell differentiation. Chinese Medicine, 16, 39.

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Assessing T Cell Proliferation and Activation by DC Stimulation

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After CD14⁺ cell isolation from PBMCs for DC preparation, autologous CD3⁺ T cells were also isolated using anti-human CD3 magnetic particles, and BD IMag™ Cell Separation Magnet. Then, the CD3⁺ T cells were labeled with CFSE by the addition of CFDA-SE (10 μM) containing RPMI 1640 medium for 15 min at 37°C. The DCs (2.5 × 10⁵ cells/ml) treated with wild-type or ΔltaS HKSG (1 × 10⁷ CFU/ml) for 16 h, were co-cultured with the CFSE-labeled CD3⁺ T cells at 1:1 ratio for 4 days. For T cell proliferation analysis, the co-cultured cells were applied to flow cytometry (FACSCalibur). For the T cell activation analysis, the cells incubated with anti-human CD25 antibody or anti-human CD4 and CD8 antibodies were subjected to flow cytometry (FACSCalibur).

Kim S.K., Im J., Ko E.B., Lee D., Seo H.S., Yun C.H, & Han S.H. (2023). Lipoteichoic acid of Streptococcus gordonii as a negative regulator of human dendritic cell activation. Frontiers in Immunology, 14, 1056949.

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Isolation and Differentiation of Human DCs

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Peripheral blood mononuclear cells (PBMCs) were isolated from human blood samples, supplied from the Korean Red Cross (Seoul, Republic of Korea), by density-gradient centrifugation with Ficoll (GE Healthcare, Uppsala, Sweden). Then, CD14⁺ monocytes were purified from PBMCs using anti-human CD14 magnetic particles and BD IMag™ Cell Separation Magnet. The cells were differentiated into DCs for 6 days in RPMI 1640 medium containing 10% FBS, 1% penicillin-streptomycin solution, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; 5 ng/ml; R&D Systems, Minneapolis, MN, USA), and interleukin-4 (IL-4) (9 ng/ml; CreaGene, Gyeonggi-Do, Republic of Korea) (23 (link)).

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Isolation and Cryopreservation of Mononuclear Cells

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Mononuclear cells (MCs) were isolated from Trima Accel leukocyte reduction system (LRS) chambers or heparinized tubes using Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation according to the manufacturer’s instructions. For long-term storage (surface screen and tissue phenotyping only), MCs were resuspended in fetal bovine serum (FBS; Omega Scientific, Inc.) with 10% DMSO, slowly cooled to −80°C, and stored in liquid nitrogen at a density of 1-5 × 10⁷ cells/mL. Cryopreserved MCs were thawed into cell culture medium (CCM; RPMI 1640 containing 10% FBS, and GlutaMAX; Thermo Fisher Scientific) supplemented with 25cU/mL benzonase (Sigma-Aldrich). and pelleted for 5cmin at 250cg. Where indicated, cells underwent magnetic lineage depletion according to the manufacturer’s instructions using BD Streptavidin Particles Plus and the BD IMag Cell Separation Magnet (BD Biosciences) with biotinylated anti-CD3 (surface screen samples) or a cocktail of biotinylated antibodies consisting of CD3, CD7, CD15, CD33, CD56, CD61, and CD235ab (other samples). The biotinylated antibody cocktail was detected by labeled anti-biotin (mass cytometry) and streptavidin (FACS) and further depleted in silico.

Glass D.R., Tsai A.G., Oliveria J.P., Hartmann F.J., Kimmey S.C., Calderon A.A., Borges L., Glass M.C., Wagar L.E., Davis M.M, & Bendall S.C. (2020). An Integrated Multi-omic Single-Cell Atlas of Human B Cell Identity. Immunity, 53(1), 217-232.e5.

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Sp1 Expression in Myeloid Cells

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Single-cell suspensions of BM cells from 8-10 week-old WT or KO mice were stained by anti-mouse Gr-1 particles (Cat No. 558111, BD Biosciences) and separated by the BD IMag Cell Separation Magnet. These harvested cells were cultured in RPMI1640 medium containing 5% FBS supplemented with 10% tumor supernatants from MC38 cells or 20 ng/mL GM-CSF for 48 h. Cells were lysed by RIPA lysis buffer, and the cell lysates were incubated on ice for 30 min and centrifuged at 13,000g, 4 °C for 15 min before the supernatants were collected. Western blot analysis was performed as previously described 10 (link). The primary antibodies included anti-Sp1 (1:1,000; Cat No. ab227383, Abcam) and anti-Actin (1:1,000; Cat No. A1978, Sigma-Aldrich).

Wu L., Xu Y., Zhao H., Zhou Y., Chen Y., Yang S., Lei J., Zhang J., Wang J., Wu Y, & Li Y. (2022). FcγRIIB potentiates differentiation of myeloid-derived suppressor cells to mediate tumor immunoescape. Theranostics, 12(2), 842-858.

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Chronic and Transient T-Cell Activation

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Freshly isolated T cells were plated in a 24‐multiwell plate (Corning) at the concentration of 1 × 10⁶ cells/well in 2 ml of complete culture medium composed of RPMI 1640 medium (Gibco) supplemented with 10% FBS, 1% Penicillin–Streptomycin (Hyclone Laboratories, Inc.) and 1% L‐glutamine (Hyclone Laboratories, Inc.). To mimic a chronic stimulation, cells were cultured for 14 days in a humified CO₂ incubator at 37°C in complete culture medium with Dynabeads® Human T‐Activator CD3/CD28 beads (ThermoFisher Scientific) (25 μl/well; bead‐to‐cell ratio of 1:1) and recombinant human interleukin‐2 (rhIL‐2; 25 U/ml; Sigma‐Aldrich). To mimic a transient stimulation, cells were cultured in the presence of CD3/CD28 beads and rhIL‐2 for 3 days and then rested in the presence of rhIL‐2 only for the remaining 11 days of culture. For both stimulation conditions, cells were collected at day 3, 7, 10, and 14. After collection, beads were magnetically removed using a BD IMag Cell Separation Magnet (BD Biosciences) prior to cell preparation for flow cytometry analysis, passaging, cryopreservation. For cell passaging, cells were resuspended in fresh complete medium for chronic or transient stimulation and replated at the same cell density as at day 0.

Corselli M., Saksena S., Nakamoto M., Lomas WE I.I.I., Taylor I, & Chattopadhyay P.K. (2021). Single cell multiomic analysis of T cell exhaustion in vitro. Cytometry, 101(1), 27-44.

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