Antibody-fluorochrome Conjugates Research Articles

Gezieltes Entfernen von Fluorochromkonjugaten nach der Zellsortierung

Flow cytometric analysis and sorting rely on fluorescent labeling with multiple antibody-fluorochrome conjugates. Currently available conjugates remain on the cells after flow sorting and thus can limit downstream applications. In contrast, REAlease® conjugates enable specific cell labeling and can be completely removed from the epitopes after the experiment; cell epitopes and fluorescence channels that were blocked through labeling become available for subsequent experiments.

Abstract 4048: REAlease® technology: Controlled release of antibody-fluorochrome conjugates for maximal flexibility in flow sorting and fluorescence microscopy applications

Abstract Multi-parameter immunofluorescent labeling is the method of choice for the flow-based sorting of targeted cell populations from heterogeneous samples. However, existing antibody-fluorochrome conjugates used for flow sorting limit the types of downstream analyses that can be applied to the isolated cells, as their continued presence on the cells blocks specific cellular epitopes and restricts the choice of downstream fluorescence detection channels. Herein, we present a new type of antibody-fluorochrome conjugates that facilitates a highly specific multi-parameter cell staining for flow sorting as well as subsequent complete release of the conjugate probes from the sample. These new conjugates rely on recombinantly engineered antibody fragments that individually possess low epitope binding affinities. A tailor-made covalent conjugation chemistry allows for their multimerization as well as fluorochrome labeling, generating fluorescent probes comparable to conventional antibody conjugates. Importantly, upon addition of a release reagent, the antibody-fluorophore conjugates can be rapidly released from the cell surface. Accordingly, previously blocked epitopes are free for re-labeling, which provides maximal flexibility for renewed epitope targeting in downstream applications. As many targeted cell populations require multi-parameter detection of several antigens for their isolation, the recombinant engineered antibodies were conjugated with different fluorochromes. The versatility of this technology is presented in multi-parameter panels enabling clear distinction and discrimination of different cell populations of different starting material, e.g. peripheral blood and tumor cells. The applicability of the new REAlease® antibody fluorochrome conjugates is also demonstrated by flow sorting of human naïve regulatory T cells. In addition, we show that the fast and efficient releasability of the new conjugates can serve as an optimal basis for cyclic immunofluorescence microscopy using a new MACSima™ imaging platform, which permits cell analysis with potentially unlimited number of parameters. Citation Format: Jennifer Pankratz, Sabine Schmachtenberg, Nicole Jansen, Marie Hansen, Susanne Krauthäuser, Ali Kinkhabwala, Karolina Schlegel, Svenja Meiler, Christiane Siewert, Alina Bartholomäus, Dmytro A. Yushchenko, Christian Dose. REAlease® technology: Controlled release of antibody-fluorochrome conjugates for maximal flexibility in flow sorting and fluorescence microscopy applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4048.

REAlease® technology: Controlled release of antibody-fluorochrome conjugates for maximal flexibility in flow sorting applications

Abstract Multi-parameter immunofluorescent labeling is the method of choice for the flow-based sorting of targeted cell populations from heterogeneous samples. However, existing antibody-fluorochrome conjugates used for flow sorting limit the types of downstream analyses that can be applied to the isolated cells, as their continued presence on the cells blocks specific cellular epitopes and restricts the choice of downstream fluorescence detection channels. Herein, we present a new type of antibody-fluorochrome conjugates that facilitates a highly specific multi-parameter cell staining for flow sorting as well as subsequent complete release of the conjugate probes from the sample. These new conjugates rely on recombinantly engineered antibody fragments that individually possess low epitope binding affinities. A tailor-made covalent conjugation chemistry allows for their multimerization as well as fluorochrome labeling, generating fluorescent probes comparable to conventional antibody conjugates. Importantly, upon addition of a release reagent, the antibody-fluorophore conjugates can be rapidly released from the cell surface. Accordingly, previously blocked epitopes are free for re-labeling, which provides maximal flexibility for renewed epitope targeting in downstream applications. The versatility of this technology is presented in multi-parameter panels enabling clear distinction and discrimination of different cell populations. The applicability of the new REAlease® antibody fluorochrome conjugates is also demonstrated by flow sorting of human naïve regulatory T cells.

Next Generation Flow (NGF) for High Sensitive Detection of Minimal Residual Disease (MRD) in Multiple Myeloma (MM)

Next Generation Flow (NGF) for High Sensitive Detection of Minimal Residual Disease (MRD) in Multiple Myeloma (MM)

Abstract 204: Understanding breast cancer resistance to chemotherapy: Characterization of cancer cell sub-populations in residual and relapsed tumors

Abstract Tumor recurrence fueled by residual tumor cells having survived chemotherapy represents the principal cause of breast cancer treatment failure. Triple-negative breast cancer (TNBC) is a heterogeneous disease at both molecular and cellular level, and the presence of different tumor cell sub-populations is likely the reason for this heterogeneity and for the incomplete response to neoadjuvant chemotherapy observed for most TNBC. To identify and isolate tumor cell sub-populations that resist to chemotherapy, we used a panel of 45 antibody fluorochrome conjugates in combination with multi-parameter flow cytometry to screen for the expression of a set of cell surface markers during the course of tumor chemotherapy. This set of markers represented both proteins involved in stem cell function and proteins known to be over-expressed in stem cells or cancer stem cell sub-populations. As a source of tumor samples, we used a panel of TNBC patient-derived xenografts (PDXs). These tumor models are known to preserve the morphology, molecular characteristics and drug response profile of the original patient tumors. We used TNBC PDX models to reproduce in vivo chemotherapy-induced tumor regression and relapse. Tumor dissociation, depletion of mouse cells and multi-parameter flow cytometry allowed us to measure the percentage of marker expression only in the fraction of human tumor cells. We found that the expression of most of markers was very heterogeneous among the different TNBC models analyzed. In residual tumors, we observed enrichment or depletion of several cell populations expressing stem and cancer stem cell markers such as CD44+ and CD133/1+. Interestingly, in the majority of relapsed tumors, the percentage of these marker-positive cells shifted back to pre-treatment levels. Moreover, the percentage of certain tumor cell sub-populations was higher in untreated chemotherapy-resistant tumors compared to untreated chemotherapy-sensitive ones. To study the properties of enriched tumor cells sub-populations, we coupled cell separation of marker-positive tumor cells to large scale molecular analysis. Microarray-based gene expression analysis of sorted tumor cells revealed cell sub-populations with up-regulated signaling pathways related to stem cell function, cell migration or EMT. Further in vivo and in vitro assays with sorted sub-populations are currently being performed to search for possible functional role of selected markers during tumor resistance to chemotherapy and initiation of tumor relapse. In summary, by coupling PDX and cell surface marker screening technologies, we have identified distinct tumor cell sub-populations which are associated with tumor resistance to chemotherapy. We believe that this approach will help gaining knowledge of tumor cell adaptation and selection in response to chemotherapy, which will ultimately lead to design tailored treatments for TNBCs. Citation Format: Andrea Aloia, Evgeniya Petrova, Olivier Deas, Sophie Banis, Enora Le Ven, Andreas Bosio, Olaf Hardt, Stefano Cairo, Jean-Gabriel Judde. Understanding breast cancer resistance to chemotherapy: Characterization of cancer cell sub-populations in residual and relapsed tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 204. doi:10.1158/1538-7445.AM2014-204

Proliferative capacity and cytokine production by cells of HIV-infected and uninfected adults with different helminth infection phenotypes in South Africa.

BackgroundIt has been suggested that the proliferative capacity of cells from individuals with HIV or both HIV and helminth infections is attenuated and cytokine production is dysregulated. This study describes peripheral blood mononuclear cell proliferation capacity and cytokine profile from individuals with HIV or both HIV and helminth infections in South Africa.MethodsForty HIV-infected and 22 HIV-uninfected participants were randomly selected and stratified into different helminth infection phenotypes by egg excretion and Ascaris lumbricoides specific –immunoglobulin-E (IgE) levels. Five day cell cultures of participants, unstimulated or stimulated with Phytohaemaglutinnin, Streptokinase, HIV-1 p24 and Ascaris lumbricoides worm antigens were stained with monoclonal antibody-fluorochrome conjugates (Ki67-FITC and CTLA-APC-4). Percentage expression of Ki67 and CTLA-4 was measured to determine cell proliferation and regulation, respectively. Culture supernatants were analysed for the expression of 13 cytokines using the Bioplex (BioRad) system. Kruskal Wallis was used to test for differences in variables between helminth infected subgroups who were either having eggs in stool and high IgE (egg+IgEhi); or eggs in stool and low IgE (egg+IgElo); or no eggs in stool and high IgE (egg-IgEhi) and those without helminth infection (egg-IgElo).ResultsIndividuals excreting eggs in stool with high serum IgE (egg+IgEhi phenotype) had potent mitogen responses but consistently produced low, but statistically non-significant antigen–specific (HIV-1 p24 (p = 0.41) and Ascaris (p = 0.19) and recall antigen (Streptokinase; p = 0.31) Ki67 responses. The group also had reduced type 1 cytokines. Individuals excreting eggs in stool with low serum IgE( egg+IgElo phenotype) had a more favourable antiviral profile, characterized by higher IFNγ, IL-2, lower IL-4 and higher IL-10 production.ConclusionThe findings suggest that dual HIV/helminth infection with egg excretion and/or high Ascaris IgE phenotye may be linked with poor proliferative capacity and deleterious cytokine profile with regards to HIV control.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-499) contains supplementary material, which is available to authorized users.

Eight colour diagnostic flow cytometry

Technological improvements are enabling flow cytometry to assess an increasing number of markers simultaneously. This presentation will describe the advantages and disadvantages of an eight colour diagnostic flow cytometry system. In the determination of lymphocyte subsets, the system enables each sample to be analysed in a single tube. In haematological malignancies, the eight colour system provides improved sensitivity in the assessment of small populations. For example, several B cell markers can be determined in the same tube as kappa and lambda, enabling detection of small monoclonal B cell populations, and distinction of monoclonal populations from reactive follicular hyperplasia. The eight colour system is very useful in assessing minimal residual disease. A greater number of markers can be determined when samples, such as CSF, are analysed in a single tube because of low cell numbers. There have also been improvements in work flow, and reductions in the cost of antibodies used in multiple tubes. Disadvantages include greater complexity in data analysis, more training for pathologists and laboratory staff, and custom preparation of antibody-fluorochrome conjugates. Implementation depends on a highly skilled laboratory scientist. On balance, the improvements in diagnosis have justified the use of an eight colour system.

Eight colour diagnostic flow cytometry

Technological improvements are enabling flow cytometry to assess an increasing number of markers simultaneously. This presentation will describe the advantages and disadvantages of an eight colour diagnostic flow cytometry system. In the determination of lymphocyte subsets, the system enables each sample to be analysed in a single tube. In haematological malignancies, the eight colour system provides improved sensitivity in the assessment of small populations. For example, several B cell markers can be determined in the same tube as kappa and lambda, enabling detection of small monoclonal B cell populations, and distinction of monoclonal populations from reactive follicular hyperplasia. The eight colour system is very useful in assessing minimal residual disease. A greater number of markers can be determined when samples, such as CSF, are analysed in a single tube because of low cell numbers. There have also been improvements in work flow, and reductions in the cost of antibodies used in multiple tubes. Disadvantages include greater complexity in data analysis, more training for pathologists and laboratory staff, and custom preparation of antibody-fluorochrome conjugates. Implementation depends on a highly skilled laboratory scientist. On balance, the improvements in diagnosis have justified the use of an eight colour system.

Measurement of T and B cell turnover with bromodeoxyuridine.

This unit describes two methods used to determine the rate of turnover of T and B cells in vivo. In each method, this is accomplished by administering 5-bromo-2'-deoxyuridine (BrdU), a thymidine analog, and measuring the rate at which cells become labeled with this DNA precursor during the S phase of the cell cycle. Cells that contain BrdU are then detected in vitro using fluorochrome-labelled anti-BrdU monoclonal antibody by flow cytometry. The unit includes an Alternate Protocol that may be useful in situations where the Basic Protocol proves incompatible with particular antibody-fluorochrome conjugates or other downstream staining procedures. Using this procedure, a wide range of antibody-fluorochrome combinations can be used.

Preliminary karyotype and chromosomal localization of ribosomal DNA sites in white spruce using fluorescence in situ hybridization

We have localized the major ribosomal DNA (rDNA) loci on metaphase chromosomes and in interphase nuclei of white spruce (2n = 24) by fluorescence in situ hybridization. Hybridization sites of the biotin-labelled rDNA probe were detected using antibody-fluorochrome conjugates and a confocal laser scanning microscope. White spruce has at least 12, and possibly as many as 14, rDNA sites, 1 site present on each of seven separate chromosome pairs. This is one of the highest numbers of rDNA loci yet reported among plant species. The position of the rDNA loci together with secondary constriction patterns permit, for the first time, all homologous pairs of white spruce chromosomes to be distinguished. We discuss the application of molecular cytogenetics in studies relating to the organization and evolution of DNA sequences within conifer genomes.