FITC Annexin V Apoptosis Detection Kit and PI/RNASE Solution.

An article published this year in “MOLECULES” using our AnnexinV-FITC Apoptosis Detection Kit and propidium iodide (PI) solution (PI/Rnase), by our customers from Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology Section, University of Pavia, Italy, in the analysis of (R)-(−)-Aloesaponol III 8-Methyl Ether from Eremurus persicus, a Novel Compound against Leishmaniosis. Congrats and Thanks.

Summary:

Leishmaniosis is a neglected tropical disease which affects several millions of people worldwide. The current drug therapies are expensive and often lack efficacy, mainly due to the development of parasite resistance. Hence, there is an urgent need for new drugs effective against Leishmania infections. As a part of our ongoing study on the phytochemical characterization and biological investigation of plants used in the traditional medicine of western and central Asia, in the present study, we focused on Eremurus persicus root extract in order to evaluate its potential in the treatment of leishmaniosis. As a result of our study, aloesaponol III 8-methyl ether (ASME) was isolated for the first time from Eremurus persicus root extract, its chemical structure elucidated by means of IR and NMR experiments and the (R) configuration assigned by optical activity measurements: chiroptical aspects were investigated with vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopies and DFT (density functional theory) quantum mechanical calculations. Concerning biological investigations, our results clearly proved that (R)-ASME inhibits Leishmania infantum promastigotes viability (IC50 73 µg/mL), inducing morphological alterations and mitochondrial potential deregulation. Moreover, it is not toxic on macrophages at the concentration tested, thus representing a promising molecule against Leishmania infections

Reference:

http://www.mdpi.com/1420-3049/22/4/519

Product link:

FITC Annexin V Apoptosis Detection Kit: http://www.immunostep.com/apoptosis-tools/3727-anxvkf-100t.html

PI/RNASE Solution: http://www.immunostep.com/solutions-buffers-chemicals/1289-pi-rnase-solution.html

BasoStep Kit

An article published this year in “METHODS MOL BIOL” comparing our BASOSTEP kit with other commercial basophil degranulation kits, by our customers from Division of Allergy and Immunology, Department of Pediatrics, The Jaffe Food Allergy Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, in the description of the methodology for performing Basophil Degranulation Assay. Congrats and Thanks.

Summary:

Basophil degranulation assay has gained importance over the last decade in both diagnosis of food allergy and evaluation of progression of immunotherapy. This assay involves the identification and quantification of the expression of CD63 molecule on basophil membrane. CD63 is a marker of multivesicular bodies that is exposed to cell membrane during the process of degranulation in which the contents of basophil granules are released. This chapter describes the methodology for performing this assay.

CD-sens is defined as the inverted value for the lowest allergen concentration giving 50% (LC50) of maximum CD63 upregulation multiplied by 100, i.e., (1/LC50 × 100, where LC50 is half maximal effective concentration). The higher the value for CD-sens, the higher is the basophil allergen sensitivity. In addition, change in mean fluorescent intensities of basophil activation marker CD203 is also useful readout.

Reference:

https://link.springer.com/protocol/10.1007%2F978-1-4939-6925-8_11

Product link:

BasoStep: http://www.immunostep.com/diagnostic-kits/2549-basostep-kit-reagent.html

DY634 Annexin V Apoptosis Detection Kit with PI.

An article published this year in “ONCOTARGET” using our Annexin V-Dy634 apoptosis detection kit, by our customers from Unidad de Diagnóstico Molecular y Celular del Cáncer, Centro de Investigación del Cáncer-IBMCC (USAL-CSIC), and IBSAL, Instituto de Investigación Biomédica de Salamanca, Spain, in the study of how the CRISPR-Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia. Congrats and Thanks.

Summary:

CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line.

CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients.

Reference:

http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path%5B%5D=15215&path%5B%5D=48659

Product link:

DY634 Annexin V Apoptosis Detection Kit with PI: http://www.immunostep.com/apoptosis-tools/3736-anxvkdy-100t.html

Cell Fixation & Permeabilization IntraCell Kit.

An article published this year in “SCIENTIFIC REPORTS” using our Cell Fixation & Permeabilization IntraCell Kit, by our customers from Division of Immunology, University Children’s Hospital and Children’s Research Center, Zurich, Switzerland, in the study of how CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development. Congrats and Thanks.

Summary:

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47phox-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47phox-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47phox-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Reference:

http://www.nature.com/articles/srep44187

Product link:

Intracell: http://www.immunostep.com/accesory-reagents/1278-intracell.html

Anti-CD41-FITC, anti-CD3-APC, anti-CD25-PE, anti-CD25-FITC, anti-CD4-APC-C750 and Stepcount tubes

An article published this year in “INTERNATIONAL JOURNAL OF CARDIOLOGY” using our anti-CD41-FITC, anti-CD3-APC, anti-CD25-PE, anti-CD25-FITC, anti-CD4-APC-C750 and our Stepcount tubes, by our customers from Department of Physiology, Faculty of Medicine and Dentistry, University of Valencia, Valencia, Spain, in the study of how Circulating microparticle subpopulations in Systemic Lupus Erythematosus are affected by disease activity. Congrats and Thanks.

Summary:

Background: Immune cells under chronic inflammation shed microparticles (MPs) that could fuel the inflammatory responses and atherosclerosis typically presented in Systemic Lupus Erythematosus (SLE). This study analyses total and subset-specific MPs from SLE patients and their possible influence on clinical features, leukocyte activation and serum cytokines.

Methods: Total MPs and derived from platelets, endothelial cells, monocytes, granulocytes and T-cells were quantified in plasma of 106 SLE patients and 33 healthy controls by flow cytometry. MP amounts were analyzed according to clinical manifestations, blood leukocyte populations and circulating cytokines (IFNα, TNFα, IL-10, BLyS, IL-17, IL-1β, CXCL10, CCL-2, CCL3, leptin). Finally, the in vitro effect of SLE-isolated MPs on the leukocyte activation status was analyzed.

Results: Total circulating MPs in SLE patients were related to increased disease duration and the presence of cardiovascular disease. Furthermore, patients displayed increased counts of MPs from platelets, monocytes and T-lymphocytes, especially in SLE patients with disease activity or with TNFαhigh-profile. Accordingly, MPs were associated with increased expression of activation markers in blood T-cells and monocytes. Finally, analyses propose a role of glucocorticoids in MPs generation and leukocyte activation since both fresh and cultured T-cells under this treatment presented higher IL-10 and CD25 production.

Conclusions: The altered profile of subset-specific SLE-MPs was influenced by the disease activity and altered status of leukocyte native cells, also associated with a TNFαhigh-profile.

Translational results: SLE patients, especially those with disease activity, displayed increased counts of MPs derived from platelets, monocytes and T-lymphocytes, which were more frequently found in TNFαhigh-type patients. The origin of such SLE-MP subsets seems to be related to the over-activated status of T-cells and monocytes characteristic of these patients. Ex vivo and in vitro analyses propose a role of glucocorticoids in the generation of circulating MPs and leukocyte activation in SLE patients.

Reference:

http://www.sciencedirect.com/science/article/pii/S0167527316337494

Product link:

anti-CD41-FITC: http://www.immunostep.com/antibodies/2654-cd41.html

anti-CD3-APC: http://www.immunostep.com/antibodies/1702-cd3.html

anti-CD25-PE: http://www.immunostep.com/antibodies/1666-cd25.html

anti-CD25-FITC: http://www.immunostep.com/antibodies/2591-cd25.html

anti-CD4-APC-C750: http://www.immunostep.com/antibodies/2292-cd4.html

Stepcount tubes: http://www.immunostep.com/diagnostic-kits/2312-stepcount.html

Propidium Iodide (PI) Solution.

An article published this year in “JOURNAL OF CELLULAR AND MOLECULAR MEDICINE” using our Propidium Iodide (PI) Solution, by our customers from Department of Physiology, Faculty of Medicine and Dentistry, University of Valencia, Valencia, Spain, in the study of how Extracellular histones disarrange vasoactive mediators release through a COX-NOS interaction in human endothelial cells. Congrats and Thanks.

Summary:

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone-mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose-dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX-1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX-2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial-dependent mediators through an up-regulation in COX-2-PGIS-PGI2 pathway which involves a COX-2-dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone-mediated pathologies.

Reference:

http://onlinelibrary.wiley.com/doi/10.1111/jcmm.13088/full

Product link:

http://www.immunostep.com/solutions-buffers-chemicals/2311-pi-staining-solution.html