Cd43 ly 48 microbeads

Manufactured by Miltenyi Biotec

CD43 (Ly-48) MicroBeads are magnetic beads coated with an antibody specific for the CD43 (Ly-48) antigen. They are designed for the isolation or depletion of CD43-positive cells from heterogeneous cell populations.

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Lab products found in correlation

Pan t cell isolation kit, by Miltenyi Biotec (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Streptomyces hyalurolyticus, by Merck Group (2 mentions) Pan t cell isolation kit 2, by Miltenyi Biotec (2 mentions) Dynabeads m 280, by Thermo Fisher Scientific (1 mentions) Naive cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions) Anti cd40 antibody, by BioLegend (1 mentions) Anti cd40, by BioLegend (1 mentions) Ova323 339 peptide, by AnaSpec (1 mentions) Interleukin 2 (il 2), by BioLegend (1 mentions) Anti pe microbeads, by Miltenyi Biotec (1 mentions) Facsdiva version 6, by BD (1 mentions) Pe cy7 anti cd40, by BioLegend (1 mentions) Anti cd86 pe, by BioLegend (1 mentions) Mem non essential amino acid, by Corning (1 mentions) Penicillin streptomycin, by Corning (1 mentions) Anti mouse igm f ab 2, by Jackson ImmunoResearch (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

CD43 microbeads (41 citations) Anti-CD43 (Ly-48) microbeads (7 citations) CD43 (Ly-48) microbead kit (2 citations)

16 protocols using cd43 ly 48 microbeads

Purification of Splenic B and T Cells

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Splenic B cells were negatively selected by staining with biotinylated anti-CD43 (clone S7) and anti-CD11b (clone M1/70) antibodies for 20 minutes on ice, washing and incubating the cells with streptavidin beads (Dynabeads M-280 Invitrogen), following the manufacturer’s instructions. A Dynal Invitrogen Beads Separator was used to purify the cells. B1-8^hi B cells were purified using biotinylated anti-CD43 (clone S7), anti-CD11b (clone M1/70) and anti-kappa (clone RMK-12) antibodies. OT-2 T cells from lymph nodes and spleen were purified using a mix of biotinylated antibodies: anti-B220 (clone RA3-6B2), anti-CD8 (clone 53-6.7), anti-NK1.1 (clone PK136), anti-CD11b, anti-GR1 (clone RB6-8C5), and anti-F4/80 (clone BM8). Splenic and lymph node B and T cells were maintained in RPMI 10% FBS supplemented with 2 mM l-glutamine, 100U/ml penicillin, 100 U/ml streptomycin, 20 µM β-mercaptoethanol, and 10 mM sodium pyruvate. Alternatively, plenic B cells and OT-2 T cells from lymph nodes were purified by negative selection using the CD43 (Ly-48) Microbeads (Miltenyi Biotec) and naive CD4 + T cell Isolation Kits (Miltenyi Biotec), respectively. The cell suspension was then loaded onto a MACS column and a MACS Separator was used to purify the cells. All antibodies used are listed in Supplementary Table 1.

Martínez-Riaño A., Delgado P., Tercero R., Barrero S., Mendoza P., Oeste C.L., Abia D., Rodríguez-Bovolenta E., Turner M, & Alarcón B. (2023). Recreation of an antigen-driven germinal center in vitro by providing B cells with phagocytic antigen. Communications Biology, 6, 437.

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Antigen-Specific T and B Cell Activation

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Negative purified naïve CD4⁺ T cells from OT-II mice were labeled by 5 μM CFSE and co-cultured with OVA_323–339 peptide (AnaSpec, Inc,) and bone marrow DCs for 3 days. The number of CFSE-labeled T cell divisions was measured by flow cytometry. Resting B cells from C57BL/6 mice were purified by CD43 (Ly-48) MicroBeads (Miltenyi Biotec). DCs (2 × 10⁴) were cocultured with 10⁵ resting B cells for 5 days in complete RPMI 1640 plus 5 μg/ml anti-CD40 (Biolegend) and IgG in the supernatant measured by ELISA (ebioscience). For B cell proliferation, CFSE labeled resting B cells were co-cultured with BM DCs for 5 days plus anti-CD40 antibody (Biolegend).

Dong G., Wang Y., Xiao W.M., Pujado S.P., Xu F., Tian C., Xiao E., Choi Y, & Graves D.T. (2015). FOXO1 Regulates Dendritic Cell Activity Through ICAM-1 and CCR7. Journal of immunology (Baltimore, Md. : 1950), 194(8), 3745-3755.

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Adoptive Transfer of T and B Cells

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T and B lymphocytes were isolated by magnetic sorting from WT or Rgs1 KD mice by negative selection using the Pan T cell isolation kit or CD43 (Ly-48) microbeads (Miltenyi Biotech.). WT and Rgs1 KD T and B cells (each 1 × 10⁷) were mixed at a 1:1 ratio in all four combinations. A total of 2 × 10⁷ cells were injected intravenously into age and gender matched NOD.SCID recipient mice. Two weeks later, mice were immunized as described previously. Mice were euthanized for analysis of lymph nodes and spleens four days later.

Caballero-Franco C, & Kissler S. (2016). The autoimmunity-associated gene RGS1 affects the frequency of T follicular helper cells. Genes and immunity, 17(4), 228-238.

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Isolation and Culture of Murine Splenic B Cells

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Naïve splenic B cells were magnetically enriched by negative selection using CD43 (Ly-48) MicroBeads (130–097-148, Miltenyi Biotec) with >95% purity following the manufacturer’s instructions. These cells were either cultured directly or further enriched or purified by magnetic bead separation or FACS, respectively. For magnetic enrichment of MZB, CD43^– B cells were stained with a CD23-PE antibody (B3B4; BioLegend), followed by magnetic separation using anti-PE MicroBeads (130–105-639; Miltenyi Biotec) leaving MZB in the flow-through and FoB in the enriched bound fractions. In some experiments, MZB (B220+CD93- CD21+CD23-) and FoB (B220+CD93- CD21-/loCD23+) were isolated by FACS. Purified B cells were cultured at 0.5 × 10⁶ cells/ml of B cell media (RPMI 1640, 10% heat-inactivated FBS, 0.05 mM 2-Mercaptoethanol, 1X nonessential amino acids, 1X penicillin/streptomycin, 10 mM HEPES, and 1 mM sodium pyruvate) with 20 µg/ml LPS (L2630; Sigma), 20 ng/ml IL-2 (575406; Biolegend), and 5 ng/ml IL-5 (581504; Biolegend). LPS and cytokines were supplemented at a half dose at 24h and 48h of ex vivo culture as described before (61 (link)).

Kania A.K., Price M.J., George-Alexander L.E., Patterson D.G., Hicks S.L., Scharer C.D, & Boss J.M. (2022). H3K27me3 demethylase UTX restrains plasma cell formation. Journal of immunology (Baltimore, Md. : 1950), 208(8), 1873-1885.

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Isolation and Characterization of Activated Mouse B Cells

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Lymphocytes were isolated from spleens as previously described [11 (link)]. Mature resting B cells were isolated by depletion of CD43⁺ cells using CD43 (Ly-48) MicroBeads according to the manufacturer’s protocol (Miltenyi Biotec). B cells (10⁶/well) were cultured in 1 ml of complete RPMI (RPMI 1640, 5 % FBS, 100 U/ml penicillin, 100 μg/ml streptomycin) in a 24-well plate with 5 ng/ml of recombinant mouse IL-4 (Gibson) and 5 μg/ml of anti-mouse IgM (Jackson ImmunoResearch Laboratories) for 24 h. B cells cultured in 1-ml complete RPMI were used as the unstimulated control. Both stimulated and unstimulated B cells were harvested and washed with FACS buffer and were incubated with 5 μg/ml Fc receptor block (anti-mouse CD16/32, Biolegend) for 5 min on ice. Cells were then stained with antibodies including APC-efluor780-anti-B220 (eBiosciences, clone HIS24, 1:200), PE-anti-CD86 (Biolegend, clone GL-1, 1:200), APC-anti-MHC class II (eBiosciences, clone M5/114.15.2, 1:1000), and PE/Cy7-anti-CD40 (Biolegend, clone 3/23, 1:50). Cells were detected using LSRII and analyzed by FACS-Diva Version 6.1.3 software (BD Biosciences).

Bracchi-Ricard V., Zha J., Smith A., Lopez-Rodriguez D.M., Bethea J.R, & Andreansky S. (2016). Chronic spinal cord injury attenuates influenza virus-specific antiviral immunity. Journal of Neuroinflammation, 13, 125.

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B Cell Isolation and Stimulation

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B cells from the spleen were enriched by MACS, collecting the negative fraction of CD43 (Ly48) Microbeads (Miltenyi Biotec). Cells were cultured in RPMI 1640 (Corning) supplemented with 10% FBS (MilliporeSigma), 1x Penicillin-Streptomycin, 1x MEM Nonessential Amino Acids (Corning), 1mM Sodium Pyruvate, 2mM GlutaMax, and 55μM 2-Mercaptoethanol (Thermo Fisher Scientific). The following concentrations were used for cell stimulation: 10μg/ml anti-mouse IgM F(ab’)2 (Jackson ImmunoResearch), 10ng/ml mouse rBaff (R&D Systems), 5μg/ml anti-mouse CD40 (1C10) and 10ng/ml mouse rIL4 (Thermo Fisher Scientific). Total BM cells were cultured in IMDM with GlutaMax (Thermo Fisher Scientific) supplemented with 10% FBS (MilliporeSigma), 1x Penicillin-Streptomycin (Corning), 55μM 2-Mercaptoethanol (Thermo Fisher Scientific) and in the presence of 10ng/ml mouse rIL7 (PrepoTech).

Ramezani-Rad P., Leung C.R., Apgar J.R, & Rickert R.C. (2020). E3 ubiquitin ligase Fbw7 regulates the survival of mature B cells. Journal of immunology (Baltimore, Md. : 1950), 204(6), 1535-1542.

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Isolation and Activation of CD43- Splenic B Cells

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CD43⁻ splenic B cells were isolated subsequent to RBC lysis (Sigma) by negative selection using CD43 (Ly-48) Microbeads (Miltenyi), and cultured with α-CD40+IL4, LPS+α-IgD and RP105 as described (3 (link)).

Park J., Welner R.S., Chan M.Y., Troppito L., Staber P.B., Tenen D.G, & Yan C.T. (2015). The DNA ligase IV syndrome R278H mutation impairs B-lymphopoiesis via error-prone non-homologous end-joining. Journal of immunology (Baltimore, Md. : 1950), 196(1), 244-255.

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HEL-Specific B Cell Adoptive Transfer

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For T cell–dependent immunization, 8- to 12-wk-old mice were injected i.v. with 10⁹ defibrinated SRBCs (TCS Bioscience) in PBS. EdU (Invitrogen) was dissolved in sterile PBS (5 mg/ml); for proliferation studies, 1 mg in a volume of 200 µl was injected i.p. 3 h before analysis; for the assessment of the kinetics of the formation of MBCs, 1.5 mg in a volume of 300 µl was injected i.p. every 12 h for 3 d (protocol adapted from Weisel et al., 2016 (link)). Adoptive transfers were performed into CD45.1⁺ or CD45.1⁺/CD45.2⁺ congenic mice. Briefly, 3 × 10⁴ HEL-binding B cells were injected i.v. into congenic recipients, followed by i.v. immunization the next day with 2 × 10⁸ SRBCs conjugated to a specific recombinant HEL protein. SRBCs in Alsever’s (TCS Bioscience) were conjugated to recombinant HEL (Sigma) or HEL^3X (R. Brink) with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Sigma) as previously described (Goodnow et al., 1988 (link)). CD45.2⁺ splenic B cells from SWHEL donor mice were purified by CD43 (Ly-48) MicroBeads (Miltenyi Biotec) depletion. For analyses and cell FACS sorting, CD45.2⁺ donor splenocytes were enriched by CD45.1-negative selection using anti-mouse biotinylated CD45.1 antibody (clone A20; eBioscience) and Anti-Biotin MicroBeads (Miltenyi Biotec).

Toboso-Navasa A., Gunawan A., Morlino G., Nakagawa R., Taddei A., Damry D., Patel Y., Chakravarty P., Janz M., Kassiotis G., Brink R., Eilers M, & Calado D.P. (2020). Restriction of memory B cell differentiation at the germinal center B cell positive selection stage. The Journal of Experimental Medicine, 217(7), e20191933.

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Single-cell B cell isolation and RNA analysis

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B cells from draining lymph node were negatively enriched with CD43 (Ly-48) MicroBeads (Miltenyi Biotec, 130-049-801), stained, single cell sorted with a FACS Aria II (BD) in 96-well plates containing 5 μL lysis buffer (TCL buffer (Qiagen, 1031576) 1% 2-β-mercaptoethanol (Sigma, M3148)) and immediately frozen at −80°C.
Single-cell RNA was purified using magnetic beads (RNAClean XP, Beckman Coulter, A63987) following the manufacturers instructions. RNA was eluted from the magnetic beads with 11 μL of a solution containing: random primers (14.5ng/ μL Invitrogen, 48190-011), tergitol (0.5% of NP-40 70% in H₂O, Sigma-Aldrich, NP40S) and RNase inhibitor (0.6 U/μL, Promega, N2615) in nuclease-free water (Qiagen), and incubated at 65°C for 3 min. cDNA was subsequently synthesized by reverse transcription with 7μL of a solution containing: SuperScript III Reverse Transcriptase, 5x buffer, DTT (SuperScript III Reverse Transcriptase, Invitrogen, 18080-044, 10,000U) dNTP (25 μM) RNase inhibitor (0.6 U/μL, Promega, N2615) in nuclease-free water (Qiagen) incubated at 1x(42°C 10min-25°C 10min-50°C 60min-94°C 5min). cDNA was stored at −20°C or immediately used for antibody gene amplification by nested polymerase chain reaction (PCR) after addition of 10 μL of nuclease-free water.

Viant C., Weymar G.H., Escolano A., Chen S., Hartweger H., Cipolla M., Gazumayan A, & Nussenzweig M.C. (2020). Antibody affinity shapes the choice between memory and germinal center B cell fates. Cell, 183(5), 1298-1311.e11.

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Splenic B Cell IL-10 Assay

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Splenic B cells were negatively purified (>95%) by magnetic separation using CD43 (Ly-48) microbeads (Miltenyi Biotec) and cultured for 72 h with 10 μg/ml mBSA, 10 μg/ml anti-CD40 (FGK4.5, BioXCell), or mBSA+anti-CD40. Supernatants from cell cultures were harvested and IL-10 was measured using mouse IL-10 DuoSet ELISA (DY417, R&D Systems) according to the manufacturer’s instructions.

Oleinika K., Rosser E.C., Matei D.E., Nistala K., Bosma A., Drozdov I, & Mauri C. (2018). CD1d-dependent immune suppression mediated by regulatory B cells through modulations of iNKT cells. Nature Communications, 9, 684.

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