Immunomagnetic beads

Manufactured by STEMCELL

Sourced in Canada, United States

Immunomagnetic beads are small, uniform, and superparamagnetic particles that can be coated with specific antibodies or ligands. They are used for the separation, isolation, and enrichment of target cells, proteins, or other biomolecules from complex biological samples.

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Lab products found in correlation

Penicillin streptomycin, by Wisent (5 mentions) Fetal bovine serum (fbs), by Avantor (5 mentions) Rpmi 1640, by Thermo Fisher Scientific (4 mentions) Dulbecco modified eagle medium (dmem), by Wisent (4 mentions) Fetal bovine serum (fbs), by Wisent (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Negative selection, by STEMCELL (1 mentions) Human embryonic kidney 293t cells, by American Type Culture Collection (1 mentions) Dulbecco s modified eagle s medium, by Wisent (1 mentions) Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions) Profiler plus kit, by Thermo Fisher Scientific (1 mentions) Rpmi 1640 complete medium, by R&D Systems (1 mentions) Recombinant interleukin 2, by R&D Systems (1 mentions) Recombinant il 1α proteins, by R&D Systems (1 mentions) Cytotox 96 non radioactive cytotoxicity assay kit, by Promega (1 mentions) Anti cd34 antibody, by STEMCELL (1 mentions) Ficoll hypaque, by Axis-Shield (1 mentions) Expi293f cells, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)

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Immunomagnetic bead selection (2 citations)

26 protocols using immunomagnetic beads

Cultivation and Activation of Human Cell Lines

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293T human embryonic kidney cells (obtained from ATCC) and TZM-bl cells (NIH AIDS Reagent Program) were maintained at 37°C under 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) (Wisent, St. Bruno, QC, Canada), supplemented with 5% fetal bovine serum (FBS) (VWR, Radnor, PA, USA) and 100 U/mL penicillin/streptomycin (Wisent). 293T cells were derived from 293 cells, into which the simian virus 40 T-antigen was inserted. TZM-bl cells were derived from HeLa cells and were engineered to stably express high levels of human CD4 and CCR5 and to contain the firefly luciferase reporter gene under the control of the HIV-1 promoter (Platt et al., 1998 (link)). Human peripheral blood mononuclear cells (PBMCs) from ten HIV-negative individuals (8 males and 2 females, unknown age) and five antiretroviral therapy (ART)-treated HIV-positive individuals (all males) obtained by leukapheresis and Ficoll-Paque density gradient isolation were cryopreserved in liquid nitrogen until further use. CD4+ T lymphocytes were purified from resting PBMCs by negative selection using immunomagnetic beads per the manufacturer’s instructions (StemCell Technologies, Vancouver, BC) and were activated with phytohemagglutinin-L (10 μg/mL) for 48 h and then maintained in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) complete medium supplemented with rIL-2 (100 U/mL).

Prévost J., Anand S.P., Rajashekar J.K., Zhu L., Richard J., Goyette G., Medjahed H., Gendron-Lepage G., Chen H.C., Chen Y., Horwitz J.A., Grunst M.W., Zolla-Pazner S., Haynes B.F., Burton D.R., Flavell R.A., Kirchhoff F., Hahn B.H., Smith AB I.I.I., Pazgier M., Nussenzweig M.C., Kumar P, & Finzi A. (2022). HIV-1 Vpu restricts Fc-mediated effector functions in vivo. Cell reports, 41(6), 111624.

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Isolation and Culture of Primary Human Cells

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HEK293T human embryonic kidney cells (obtained from ATCC) were grown as previously described (Richard et al., 2015 (link); Veillette et al., 2014b (link)). HEK293T cells were derived from female human embryonic kidney cells, into which the simian virus 40 T-antigen was inserted. Primary human PBMCs, and CD4+ T cells were isolated, activated and cultured as previously described (Richard et al., 2015 (link); Veillette et al., 2014b (link)). Briefly, PBMCs were obtained by leukapheresis from 7 HIV-uninfected healthy adult (6 males, 1 female) donors. CD4+ T lymphocytes were purified from resting PBMCs by a negative selection using immunomagnetic beads per the manufacturer’s instructions (StemCell Technologies, Vancouver, BC). CD4+ T lymphocytes were activated with phytohemagglutinin-L (10 μg/ mL) for 48 hours and then maintained in RPMI 1640 complete medium supplemented with rIL-2 (100 U/mL). Primary CD4 T cells were isolated from PBMCs obtained from 7 viremic untreated HIV-1-infected individuals (5 males, 2 females). Purified CD4+ T cells were activated with PHA-L at 10 μg /ml for 36 hours and then cultured for 6 days in RPMI-1640 complete medium supplemented with rIL-2 (100U/ml).

Alsahafi N., Bakouche N., Kazemi M., Richard J., Ding S., Bhattacharyya S., Das D., Anand S.P., Prévost J., Tolbert W.D., Lu H., Medjahed H., Gendron-Lepage G., Delgado G.G., Kirk S., Melillo B., Mothes W., Sodroski J., Smith AB I.I.I., Kaufmann D.E., Wu X., Pazgier M., Rouiller I., Finzi A, & Munro J.B. (2019). An Asymmetric Opening of HIV-1 Envelope Mediates Antibody-Dependent Cellular Cytotoxicity. Cell host & microbe, 25(4), 578-587.e5.

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Isolation and Purification of PBMC

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Peripheral blood mononuclear cells (PBMC) were isolated from blood by density gradient centrifugation (Ficoll-Paque; Pharmacia, Uppsala, Sweden) and cryopreserved in 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) with 90% fetal bovine serum (FBS; Wisent, Inc. St. Bruno, QC, Canada). CD4 T cells were isolated from thawed PBMC by positive selection using immunomagnetic beads (STEMCELL Technologies, Inc. Vancouver, BC, Canada). The purity of the CD4 cell population was verified by flow cytometry (average 95.3%). NK cells were isolated from thawed PBMC by negative selection (STEMCELL Technologies, Inc.) and yielded an average purity of 97.2%.

Song R., Lisovsky I., Lebouché B., Routy J.P., Bruneau J, & Bernard N.F. (2014). HIV Protective KIR3DL1/S1-HLA-B Genotypes Influence NK Cell-Mediated Inhibition of HIV Replication in Autologous CD4 Targets. PLoS Pathogens, 10(1), e1003867.

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Culturing and Activating Primary CD4+ T Cells

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First, 293T human embryonic kidney cells (obtained from ATCC, Manassas, VA, USA) were cultured at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle’s medium (Wisent) containing 5% fetal bovine serum (VWR, Radnor, PA, USA) and 100 µg/mL of penicillin-streptomycin (Wisent, St. Bruno, QC, Canada). Primary CD4+ T lymphocytes were purified from resting PBMCs by negative selection and activated as previously described [12 (link),13 (link)]. Briefly, PBMC were obtained by leukapheresis. CD4+ T lymphocytes were purified using immunomagnetic beads as per the manufacturer’s instructions (StemCell Technologies, Vancouver, BC, Canada). CD4+ T lymphocytes were activated with phytohemagglutinin-L (PHA-L; 10 µg/mL) for 48 h and then maintained in RPMI 1640 (Gibco, Waltham, MA, USA) complete medium supplemented with rIL-2 (100 U/mL).

Anand S.P., Prévost J., Descôteaux-Dinelle J., Richard J., Nguyen D.N., Medjahed H., Chen H.C., Smith AB I.I.I., Pazgier M, & Finzi A. (2021). HIV-1 Envelope Glycoprotein Cell Surface Localization Is Associated with Antibody-Induced Internalization. Viruses, 13(10), 1953.

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Culturing Human Cell Lines and Primary Cells

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HEK293T human embryonic kidney cells (obtained from the ATCC) were grown as previously described (3 (link), 9 (link)). TZM-bl cells were cultured as we described previously (77 (link)). Primary human PBMCs and CD4⁺ T cells were isolated, activated, and cultured as previously described (3 (link), 9 (link)). Briefly, PBMCs were obtained by leukapheresis, and CD4⁺ T lymphocytes were purified from resting PBMCs by negative selection using immunomagnetic beads (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions and were activated with phytohemagglutinin-L (10 μg/ml) for 48 h and then maintained in RPMI 1640 complete medium supplemented with recombinant interleukin 2 (rIL-2) (100 U/ml).

Prévost J., Richard J., Medjahed H., Alexander A., Jones J., Kappes J.C., Ochsenbauer C, & Finzi A. (2018). Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses. Journal of Virology, 92(13), e00484-18.

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Isolation and culture of HUVECs, CD34+ cells, and primary MKs

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HUVECs were purchased from the cell bank of the Chinese Academy of Sciences (CAS, Shanghai) and cultured in Minimum Essential Medium (Gibco, USA) with 10% fetal bovine serum. CD34⁺ cells and human primary MKs were obtained from human umbilical cord blood using immunomagnetic beads (Stem Cell Technologies, Canada) as previously described [16 (link)]. The CD34⁺ cells were then seeded in 24-well plates with a density of 4 × 10⁴/well, and were further cultured in serum-free medium (Stem Cell Technologies, Canada) containing 20 ng/ml recombinant human thrombopoietin (rhTPO) and 1% penicillin/streptomycin. The use of human umbilical cord blood was approved by the Human Medical Ethics Committee of the Third Military Medical University.

Chen F., Shen M., Zeng D., Wang C., Wang S., Chen S., Tang Y., Hu M., Chen M., Su Y., Ran X., Xu Y, & Wang J. (2017). Effect of radiation-induced endothelial cell injury on platelet regeneration by megakaryocytes. Journal of Radiation Research, 58(4), 456-463.

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Chimerism Determination Post-Transplantation

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Post-transplantation D30 (range, 20-50) and 100 (range, 80-120) chimerism was determined using recipient PB or BM samples collected in EDTA. “Total donor cell” chimerism was performed on buffy coat leukocytes. “T-cell” chimerism was on Ficoll Hypaque separated lymphocytes from which purified CD3+ T-cells were isolated using immunomagnetic beads (Stem Cell Technologies Inc.). Pretransplantation PB samples were used to determine the donor and recipient genotypes based on 9 CODIS Short Tandem Repeat loci using the Applied Biosystems Profiler Plus® kit with the ABI 3130 capillary genetic analyzer to determine the alleles at each locus. Informative alleles unique to either donor or recipient were used to calculate % donor chimerism at each locus using the median peak intensity of amplicons attributable to donor divided by the sum of all amplicons at that locus. In cases where there were only unique recipient amplicons, % donor chimerism was calculated as (100 – % recipient chimerism). 95% confidence interval for chimerism was ±5%.

Koreth J., Kim H.T., Nikiforow S., Milford E.L., Armand P., Cutler C., Glotzbecker B., Ho V.T., Antin J.H., Soiffer R.J., Ritz J, & Alyea EP I.I.I. (2014). Donor Chimerism Early after Reduced-intensity Conditioning Hematopoietic Stem Cell Transplantation Predicts Relapse and Survival. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 20(10), 1516-1521.

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Isolation and Activation of Immune Cells

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HEK293T human embryonic kidney cells and P815 mouse lymphoblast-like mastocytoma cells (obtained from ATCC) were grown as previously described (39 (link), 65 (link)). Primary human peripheral blood mononuclear cells (PBMCs), CD4⁺ T cells, and NK cells were isolated, activated, and cultured as previously described (66 (link), 67 (link)). Briefly, PBMCs were obtained by Ficoll density gradient from whole-blood samples obtained from 9 different HIV-1-negative donors. CD4⁺ T lymphocytes and NK cells were purified from resting PBMCs by negative selection using immunomagnetic beads per the manufacturer’s instructions (StemCell Technologies, Vancouver, BC, Canada). CD4⁺ T cells were activated with phytohemagglutinin-L (10 μg/ml) for 48 h and then maintained in RPMI 1640 complete medium supplemented with recombinant interleukin-2 (100 U/ml; R&D Systems) (see Fig. S1 in the supplemental material). NK cells were isolated and rested overnight in RPMI 1640 complete medium on the day prior to the redirection and killing assays.

Prévost J., Pickering S., Mumby M.J., Medjahed H., Gendron-Lepage G., Delgado G.G., Dirk B.S., Dikeakos J.D., Stürzel C.M., Sauter D., Kirchhoff F., Bibollet-Ruche F., Hahn B.H., Dubé M., Kaufmann D.E., Neil S.J., Finzi A, & Richard J. (2019). Upregulation of BST-2 by Type I Interferons Reduces the Capacity of Vpu To Protect HIV-1-Infected Cells from NK Cell Responses. mBio, 10(3), e01113-19.

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Culturing HEK293T, TZM-bl, and Primary Human T Cells

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HEK293T human embryonic kidney cells and TZM-bl cells obtained from ATCC were grown as previously described [7 (link),17 (link)]. Primary human PBMCs and CD4+ T cells were isolated, activated, and cultured as previously described [7 (link),17 (link)]. Briefly, PBMC were obtained by leukapheresis. CD4+ T lymphocytes were then purified from resting PBMCs via negative selection using immunomagnetic beads per the manufacturer’s instructions (StemCell Technologies, Vancouver, BC, Canada). CD4+ T lymphocytes were activated with phytohemagglutinin-L (10 µg/mL) for 48 h and then maintained in RPMI 1640 complete medium supplemented with rIL-2 (100 U/mL).

Ding S., Tolbert W.D., Zhu H., Lee D., Marchitto L., Higgins T., Zhao X., Nguyen D., Sherburn R., Richard J., Gendron-Lepage G., Medjahed H., Mohammadi M., Abrams C., Pazgier M., Smith AB I.I.I, & Finzi A. (2023). Piperidine CD4-Mimetic Compounds Expose Vulnerable Env Epitopes Sensitizing HIV-1-Infected Cells to ADCC. Viruses, 15(5), 1185.

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Cytotoxicity Assay for CD8+ T Cells

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The CytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, WI) was used to measure the cytotoxic activity of CD8⁺ T cells against tumor cells. After 1 week of tumor inoculation, splenocytes were harvested from tumor-bearing mice and stimulated with hepa1-6 cell lysates plus rhIL-2 (100 U/mL) for 4 days. For assays of examining the in vitro function of IL-1α, splenocytes were harvested from mice and stimulated with hepa1-6 cell lysates plus rhIL-2 (100 U/mL) with or without 25 ng/ml recombinant IL-1α proteins (R&D Systems, Minneapolis, MN) for 4 days. CD8⁺ T cells were sorted from splenocytes by negative selection using immunomagnetic beads (StemCell Technologies, Vancouver, BC) according to the manufacturer’s protocol. Then the cytotoxicity assay was performed using hepa1-6 cells as the target cells.

Lin D., Mei Y., Lei L., Binte Hanafi Z., Jin Z., Liu Y., Song Y., Zhang Y., Hu B., Liu C., Lu J, & Liu H. (2022). Immune suppressive function of IL-1α release in the tumor microenvironment regulated by calpain 1. Oncoimmunology, 11(1), 2088467.

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