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Cd43 microbead

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CD43 microbeads are a type of magnetic separation reagent used for the enrichment or depletion of CD43-positive cells from heterogeneous cell populations. They are composed of small, superparamagnetic particles coated with antibodies specific for the CD43 surface antigen.

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Lab products found in correlation

Interleukin 4 (il 4), by Thermo Fisher Scientific (6 mentions) Lipopolysaccharides (lps), by Merck Group (5 mentions) Interleukin 4 (il 4), by R&D Systems (4 mentions) Anti igm f ab 2, by Jackson ImmunoResearch (4 mentions) Mem non essential amino acid, by Corning (3 mentions) Glutamax, by Thermo Fisher Scientific (3 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (3 mentions) Rpmi 1640, by Corning (3 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions) Anti biotin microbead, by Miltenyi Biotec (2 mentions) Mouse ril 4, by Thermo Fisher Scientific (2 mentions) Goat anti mouse igm f ab 2, by Jackson ImmunoResearch (2 mentions) Cd43 microbeads, by Thermo Fisher Scientific (2 mentions) Cd38 90 biotin, by Thermo Fisher Scientific (2 mentions) Cd11c n418 biotin, by Thermo Fisher Scientific (2 mentions) Goat anti mouse igg f ab 2, by Jackson ImmunoResearch (2 mentions) Goat anti mouse κ f ab 2, by Southern Biotech (2 mentions) Mouse rbaff, by R&D Systems (2 mentions) Rat anti mouse cd40 1c10, by Thermo Fisher Scientific (2 mentions) Ifn γ, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

Anti-CD43 MicroBeads (35 citations) CD43 (Ly-48) MicroBeads (16 citations) Anti-CD43 (Ly-48) microbeads (7 citations) CD43-microbead depletion (5 citations) CD43 MACS Microbeads (5 citations) CD43-magnetic microbeads (3 citations) Anti-CD43 magnetic microbeads (3 citations) CD43 (Ly-48) microbead kit (2 citations) CD43 microbeads beads (2 citations) Anti-mouse CD43 Miltenyi microbeads (2 citations)

41 protocols using cd43 microbead

1

B Cell Proliferation Assay

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Naive B cells have been isolated from 3 polled spleens of sex-matched 12-weeks-old littermate mice using CD43 microbead (Miltenyi Biotec) negative selection and were cultured in RPMI 1640 containing 15% FBS. B cells were activated by the addition of 20 µg/ml LPS (Sigma) and IL-4 (R&D Systems) at 20 ng/ml to the culture media. Both primary murine B-cells and human MM cell lines have been stained with 5 μM CFSE according to manufacturer’s instructions and cultured for 72 h or 48 and 72 h, respectively. To calculate the rate of CFSE fluorescent signal dilution autofluorescence, defined as the signal from unstained samples, have been subtracted from the fluorescence of the stained cells and initial (0 h) values have been divided by ones measured at 48 and 72 h. The results were presented as relative to the control transfected with an empty vector.
Mroczek S., Chlebowska J., Kuliński T.M., Gewartowska O., Gruchota J., Cysewski D., Liudkovska V., Borsuk E., Nowis D, & Dziembowski A. (2017). The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma. Nature Communications, 8, 619.
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2

Splenic B cell CSR culture

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Splenic B cells from sex-matched littermate mice were prepared using CD43 microbead (Miltenyi Biotec) negative selection and cultured in RPMI 1640 containing 15% FBS. Ex vivo CSR cultures using the ROSA26CreERt2 allele were cultured for 16 h with 100 nM4-hydroxytamoxifen (Sigma) and 20 μg ml−1 LPS (Sigma) followed by the addition of IL-4 (R&D Systems) at 20 ng ml−1, and cultured for an additional 72 h. CSR culture conditions using the AicdaCre allele were identical with the exception that 4-hydroxytamoxifen was not used. Cells were stained using fluorescent antibodies against B220 and IgG1 (BD Biosciences). Data were acquired on a FACSAria cell sorter (BD Biosciences) and analysed using FloJo software (Tree Star). Antibodies used for western blot purposes: Exosc3 (Santa Cruz Biotechnology), actin (Abcam) and goat anti-rabbit IgG HRP (Jackson ImmunoResearch). All experiments involving mice were performed with known genotypes and therefore performed unblinded. Biological replicates involve B cells isolated from separate mice.
Pefanis E., Wang J., Rothschild G., Lim J., Chao J., Rabadan R., Economides A.N, & Basu U. (2014). Noncoding RNA transcription targets AID to divergently transcribed loci in B cells. Nature, 514(7522), 389-393.
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3

Splenic B Cell Activation Assay

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Splenocytes were collected, red blood cells were lysed and cells were CD43-depleted using CD43 microbeads (Miltenyi Biotec). Splenic B lymphocytes were cultured for 3 days in RPMI containing 10% FCS with LPS (1μg/mL, B4 Invivogen) or LPS + IL4 (20ng/mL, Peprotech). Cells and supernatants were recovered for LAM-HTGTS experiments and ELISA.
Le Noir S., Bonaud A., Hervé B., Baylet A., Boyer F., Lecardeur S., Oruc Z., Sirac C, & Cogné M. (2021). IgH 3’ regulatory region increases ectopic class switch recombination. PLoS Genetics, 17(2), e1009288.
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4

Evaluating B Cell Viability and Apoptosis

Cited 2 times
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To analyze B cell viability in vitro, B cells were purified from the spleens using CD43 microbeads (Miltenyi Biotec) as described by the manufacturer. The purified B cells were either left untreated in the medium (RPMI with 10% FBS) or stimulated with 10 μg/ml of anti-IgM F(ab′)2 (Jackson ImmunoResearch) or lipopolysaccharide (LPS, 5 μg/ml) for the indicated times. The viability of cells was measured by trypan blue exclusion assay (Invitrogen).
For analysis of apoptosis, splenocytes were stimulated with 10 μg/ml of anti-IgM F(ab′)2 (Jackson ImmunoResearch) for 30 hours. The cells were stained with anti-B220 antibody, and the apoptotic B cells (B220+ gated cells) were assayed using the Annexin V apoptosis detection kit (BD Bioscience) as previously described [30 (link), 34 (link)].
In vitro proliferation assay was performed as previously described [30 (link), 42 (link), 43 ]. Briefly, splenocytes (5 × 106) were stained with CFSE (Molecular Probes, Eugene, OR) at a final concentration of 2 μM for 10 minutes. The cells were washed and then treated either with medium (RPMI with 10% FBS) alone or medium plus anti-IgM F(ab′)2 or LPS for 30 hours. The flow cytometry analysis was carried out on B220+ gated cells [26 (link), 30 (link)].
Oleksyn D., Zhao J., Vosoughi A., Zhao J., Misra R., Pentland A., Ryan D., Anolik J., Ritchlin C., Looney J., Anandarajah A., Schwartz G., Calvi L., Georger M., Mohan C., Sanz I, & Chen L. (2017). PKK deficiency in B cells prevents lupus development in Sle lupus mice. Immunology letters, 185, 1-11.
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5

Isolation and Culture of Germinal Center B Cells

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B splenocytes were MACS-purified, collecting the negative fraction of CD43 Microbeads (Miltenyi Biotec). GC B cells were MACS-purified, collecting the negative fraction of CD43 Microbeads, CD38 (90) Biotin (Thermo Fisher Scientific), CD11c (N418) Biotin (Thermo Fisher Scientific) and anti-Biotin Microbeads (Miltenyi Biotec). Cells were cultured in RPMI 1640 (Corning) supplemented with 10% FBS (MilliporeSigma and Thermo Fisher Scientific), 1x Penicillin-Streptomycin, 1x MEM Nonessential Amino Acids (Corning), 1mM Sodium Pyruvate, 2mM GlutaMax, and 55 μM 2-Mercaptoethanol (Thermo Fisher Scientific). Cells were stimulated with the following reagents: 10μg/ml goat anti-mouse IgM F(ab’)2, 10μg/ml goat anti-mouse IgG F(ab’)2 (Jackson ImmunoResearch Labs), 10μg/ml goat anti-mouse κ F(ab’)2 (SouthernBiotech), 10ng/ml mouse rBaff (R&D Systems), 5μg/ml rat anti-mouse CD40 (1C10) and 10ng/ml mouse rIL-4 (Thermo Fisher Scientific). For in vitro-derived GC B cell cultures, B splenocytes were cultured on 40LB cells (3T3 fibroblasts expressing CD40L and secreting BAFF) with addition of 1ng/ml mouse rIL-4.
Ramezani-Rad P., Chen C., Zhu Z, & Rickert R.C. (2020). Cyclin D3 Governs Clonal Expansion of Dark Zone Germinal Center B Cells. Cell reports, 33(7), 108403.
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6

B Cell Stimulation and Immunization

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Reagents for primary B cell stimulation include CD43-microbeads (Miltenyi Biotec), LPS (50 μg/ml; Sigma-Aldrich), IFN-γ (100 ng/ml; Peprotech) and IL-4 (5 ng/ml; Peprotech). NP-CGG (75–100 μg/mouse, Biosearch Technologies Inc.) was used for immunization experiments.
Robert I., Gaudot L., Rogier M., Heyer V., Noll A., Dantzer F, & Reina-San-Martin B. (2015). Parp3 Negatively Regulates Immunoglobulin Class Switch Recombination. PLoS Genetics, 11(5), e1005240.
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7

B Cell Activation and Differentiation

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Spleens were harvested at day 8 post-immunization. Splenocytes were collected, red blood cells were lysed and cells were CD43-depleted using CD43 microbeads (Miltenyi Biotec). Single-cell suspensions of CD43- B splenocytes from wt, and LSRµKI mice were cultured 3 days at 1x106 cells/ml in RPMI 1640 with 10% fetal calf serum, 5μg/ml LPS with or without, 20ng/ml IL-4, 2ng/ml TGFβ and 2 ng/ml INFγ (PeproTech, Rocky Hill, NJ), anti-CD40 antibody (2.5μg/ml) (R&D systems) and anti-κ light chain antibody (2.5µg/ml, Southern Biotechnology) for BCR cross-linking. Culture samples were harvested at day 4 for RNA extraction. Culture supernatants were then recovered and stored at -20°C until used.
Denis-Lagache N., Oblet C., Marchiol T., Baylet A., Têteau O., Dalloul I., Dalloul Z., Zawil L., Dézé O., Cook-Moreau J., Saintamand A., Boutouil H., Khamlichi A.A., Carrion C., Péron S., Le Noir S., Laffleur B, & Cogné M. (2023). Attempts to evaluate locus suicide recombination and its potential role in B cell negative selection in the mouse. Frontiers in Immunology, 14, 1155906.
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8

Generating Murine B-Cell Plasmablasts

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B cells were isolated from the spleens of Ptpn6+/+ and Ptpn6f/f mice by negative selection using CD43 microbeads (Miltenyi Biotec). After three washes with HyQADCF-Mab medium (Hyclone), 1 × 107 cells per ml were incubated for 45 min at 37 °C with TAT-Cre at a concentration of 35 mg ml−1 (ref. 66 (link)). Cells were again washed twice with RPMI medium plus 10% FBS, and cultured for 3 days at a density of 2 × 106 cells per ml with LPS (20 μg ml−1) to induce plasmablast in vitro.
Li Y.F., Xu S., Ou X, & Lam K.P. (2014). Shp1 signalling is required to establish the long-lived bone marrow plasma cell pool. Nature Communications, 5, 4273.
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9

Isolation and Purification of Murine B and T Cells

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B cells were purified from the spleens of IL-10−/− or C57BL/6 mice by negative selection using CD43 microbeads (Miltenyi Biotech, 130-049-801) according to manufacturer’s instructions. Purified splenic B cells were stained with anti-CD21, anti-CD24 anti-CD23. DAPI was added to exclude dead cells. Cells were sorted by BD FACSAria II (BD Biosciences). CD4+ T cells were isolated from the spleens of C57BL/6 mice using Invitrogen Dynabeads® Untouched™ Mouse CD4 Cells kits, according to manufacturer’s instructions (Invitrogen, 11416D).
Alhabbab R., Blair P., Elgueta R., Stolarczyk E., Marks E., Becker P.D., Ratnasothy K., Smyth L., Safinia N., Sharif-Paghaleh E., O’Connell S., Noelle R.J., Lord G.M., Howard J.K., Spencer J., Lechler R.I, & Lombardi G. (2015). Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model. Scientific Reports, 5, 11554.
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10

Isolated Naive B Cell Stimulation and qRT-PCR

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WT splenic naive B cells were isolated using depletion with CD43 Micro-Beads (Miltenyi Biotec) and stimulated with anti-CD40 (1 µg/ml; HM40-3; Invitrogen) or polyclonal anti-IgM F(ab′)2 (1 µg/ml; Jackson ImmunoResearch) antibody alone or in combination with IL-4 (20 ng/ml, Peprotech) for the indicated time. Total RNA was isolated from cultured B cells using the Quick-RNA Microprep Kit (Zymo Research). cDNA was synthesized with the RevertAid RT Kit (Thermo Fisher Scientific), and qRT-PCR was performed on a CFX96-C1000 Thermo Cycler (Bio-Rad) using the SensiFAST SYBR No-ROX Kit (Bioline; Meridian Bioscience). Hprt1 was used as a housekeeping gene, and the standard curve method was used for quantification. The following primers were used: Bhlhe40-F, 5′-CTC​CTA​CCC​GAA​CAT​CTC​AAA​C-3′, Bhlhe40-R, 5′-CCA​GAA​CCA​CTG​CTT​TTT​CC-3′, Hprt1-F, 5′-AGT​GTT​GGA​TAC​AGG​CCA​GAC-3′, and Hprt1-R, 5′-CGT​GAT​TCA​AAT​CCC​TGA​AGT-3′.
Rauschmeier R., Reinhardt A., Gustafsson C., Glaros V., Artemov A.V., Dunst J., Taneja R., Adameyko I., Månsson R., Busslinger M, & Kreslavsky T. (2021). Bhlhe40 function in activated B and TFH cells restrains the GC reaction and prevents lymphomagenesis. The Journal of Experimental Medicine, 219(2), e20211406.
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