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(A) Starting with immunized BALB/c mouse splenocytes, the CD138+ cell content of the isolated fraction is typically 81.5 ± 4.9% (mean ± SD). In the above example, the purities of the start and final isolated fractions are 4.6% and 83.3%, respectively.
(B) Starting with immunized BALB/c mouse splenocytes, the plasma cell (CD138+CD267 (TACI)+) content is typically 68.5 ± 11.3% (mean ± SD). In the above example, the purities of the start and final isolated fractions are 1.3% and 70.2%, respectively.
(C) Starting with naïve C57BL/6 mouse splenocytes, the CD138+ cell content of the isolated fraction is typically 78.3 ± 5.7% (mean ± SD). In the above example, the purities of the start and final isolated fractions are 5.0% and 79.5%, respectively.
(D) Starting with naïve C57BL/6 mouse splenocytes, the plasma cell (CD138+CD267 (TACI)+) content is typically 50.8 ± 10.0 % (mean ± SD). In the above example, the purities of the start and final isolated fractions are 0.4% and 56.3%, respectively.
(E) Isolated CD138+ cells or total splenocytes from mice immunized with various antigens were fused with Sp2/0 mouse myeloma cells and plated in semi-solid medium using ClonaCell™-HY Hybridoma Kit (Catalog #03800). The % antigen-specific hit rate was determined by ELISA. The % antigen-specific hit rates for total splenocytes and CD138+ cells were 4.1 ± 1.6% and 28.8 ± 11.0% (mean ± SD), respectively.
(F) Isolated CD138+ cells or total splenocytes from mice immunized with various antigens were fused with Sp2/0 mouse myeloma cells and plated in semi-solid medium using ClonaCell™-HY Hybridoma Kit (Catalog #03800). The % antibody-secreting hybridomas was determined by ELISA. The % antibody-secreting hybridomas for total splenocytes and CD138+ cells were 33.0 ± 8.7% and 99.3 ± 0.6% (mean ± SD), respectively.
Structural stabilization of the intrinsically disordered SARS-CoV-2 N by binding to RNA sequences engineered from the viral genome fragment
Nature Communications 2025 Jul
Abstract
The nucleocapsid N is one of four structural proteins of the coronaviruses. Its essential role in genome encapsidation makes it a critical therapeutic target for COVID-19 and related diseases. However, the inherent disorder of full-length N hampers its structural analysis. Here, we describe a stepwise method using viral-derived RNAs to stabilize SARS-CoV-2 N for EM analysis. We identify pieces of RNA from the SARS-CoV-2 genome that promote the formation of structurally homogeneous N dimers, intermediates of assembly, and filamentous capsid-like structures. Building on these results, we engineer a symmetric RNA to stabilize N protein dimers, the building block of high-order assemblies, for EM studies. We combine domain-specific monoclonal antibodies against N with chemical cross-linking mass spectrometry to validate the spatial arrangement of the N domains within the dimer. Additionally, our cryo-EM analysis reveals novel antigenic sites on the N protein. Our findings provide insights into N protein´s architectural and antigenic principles, which can guide design of pan-coronavirus therapeutics. The authors stabilize the SARS-CoV-2 nucleocapsid (N) dimer assembly using a short RNA and chemical crosslinker for EM analysis, revealing its domain arrangement and antigenic sites to advance understanding and guide pan-coronavirus therapeutic design.
Preclinical development of an immunoassay for the detection of TREM2: a new biomarker for Alzheimer’s disease
Scientific Reports 2025 Jul
Abstract
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein. The combination of biomarkers is crucial for AD diagnosis. The triggering receptor expressed on myeloid cells 2 (TREM2), a receptor expressed on microglia, is important in AD pathogenesis. Impairment of TREM2 function aggravates the toxic effects of amyloid plaques, and its activation has been shown to reduce Aβ burden and memory deficits. Increased levels of soluble TREM2 (sTREM2) in blood and cerebrospinal fluid is associated with AD. Therefore, TREM2 could serve as a non-invasive biomarker for AD. In this study, we developed a preclinical immunoassay to detect TREM2 for AD diagnosis. Highly sensitive and specific TREM2 antibodies were produced using the hybridoma technique. The three optimized immunoassays exhibited lower limit of quantitation (LLOQ) of 0.474, 0.807, and 0.415 ng/mL, respectively. These preclinical immunoassays showed high sensitivity and specificity. The sandwich enzyme-linked immunosorbent assay (ELISA) could potentially be used for AD diagnosis.
Sensory neurons regulate stimulus-dependent humoral immunity in mouse models of bacterial infection and asthma
Nature Communications 2024 Oct
Abstract
Sensory neurons sense pathogenic infiltration to drive innate immune responses, but their role in humoral immunity is unclear. Here, using mouse models of Streptococcus pneumoniae infection and Alternaria alternata asthma, we show that sensory neurons are required for B cell recruitment and antibody production. In response to S. pneumoniae, sensory neuron depletion increases bacterial burden and reduces B cell numbers, IgG release, and neutrophil stimulation. Meanwhile, during A. alternata-induced airway inflammation, sensory neuron depletion decreases B cell population sizes, IgE levels, and asthmatic characteristics. Mechanistically, during bacterial infection, sensory neurons preferentially release vasoactive intestinal polypeptide (VIP). In response to asthma, sensory neurons release substance P. Administration of VIP into sensory neuron-depleted mice suppresses bacterial burden, while VIPR1 deficiency increases infection. Similarly, exogenous substance P delivery aggravates asthma in sensory neuron-depleted mice, while substance P deficiency ameliorates asthma. Our data, thus demonstrate that sensory neurons release select neuropeptides which target B cells dependent on the immunogen. Sensory neurons may regulate innate immune cells, but their roles in humoral immunity is still unclear. Here the authors show that bacterial infection and asthma induction induce sensory neuron production of distinct neurotransmitters to dampen B cell responses but differentially target IgG and IgE, respectively, to specifically modulate the symptoms.
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