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T Cell Depletion of Allogeneic Human Bone Marrow Grafts by Soybean Lectin Agglutination and Either Sheep Red Blood Cell Rosetting or Adherence on the CD5/CD8 Cellector™
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
Nancy H. Collins, Nancy A. Kernan, Sharon A. Bleau, Richard J. O’Reilly
The number of T cells remaining after either rosetting, or the alternative methods outlined in the following section, is below that which can be accurately quantitated by rosetting or PHA stimulation. Therefore, we developed a limiting dilution cloning technique for the outgrowth of T cells in the SBA−E− population. This requires only 1 × 106 bone marrow cells and correlates to the occurrence of GvHD.19 Advances in flow cytometry technology since the development of the limiting dilution assay now allow the detection of the residual T cells, such that the examination of the SBA−E− graft is possible before transplantation.
Production of a biosimilar version of aflibercept to improve VEGF blocker cytotoxicity on endothelial cells
Published in Growth Factors, 2023
Amin Ramezani, Mohammadrasul Zareinejad, Elham Mahmoudi Maymand, Elina Kaviani, Abbas Ghaderi
Stable cell selection was conducted based on a three-phase selection strategy, using CD FortiCHO Medium (Invitrogen, Carlsbad, CA) supplemented with anti-clumping agent (Gibco, Carlsbad, CA) (at a dilution of 1:100), and increasing concentrations of puromycin (Gibco, Carlsbad, CA) and methotrexate (MTX) (Sigma, St. Louis, MO) (Ramezani and Ghaderi 2018). After a three-phase selection scheme and the creation of four stable cell pools with high production capacity, single-cell clone isolation was performed by the limiting dilution cloning (LDC) method. The stability of selected clones was assessed by culturing clones on Dynamis™ medium (Gibco, Carlsbad, CA) in shake flasks over approximately 40 generations. Purification was performed from cell culture supernatant using the AKTA pure 150 FPLC system (GE Healthcare, Stockholm, Sweden) and MabSelect™ SuRe™ (Cytiva, Marlborough, MA), and Sephadex® G-25 resins (Cytiva, Marlborough, MA).
Functional genetic evaluation of DNA house-cleaning enzymes in the malaria parasite: dUTPase and Ap4AH are essential in Plasmodium berghei but ITPase and NDH are dispensable
Published in Expert Opinion on Therapeutic Targets, 2019
Hirdesh Kumar, Jessica Kehrer, Mirko Singer, Miriam Reinig, Jorge M. Santos, Gunnar R. Mair, Friedrich Frischknecht
Establishing a clonal ap4ah(-) mutant also failed although one transfection attempt out of four had resulted in the integration of the plasmid into the genome (Supplementary Figure 15A). The isolation of an isogenic, mutant clone however proved unsuccessful in two attempts (n = 16); limiting dilution cloning produced either non-infected mice or animals infected with wild type parasites only. In a third cloning attempt, we injected 5 mice with 10 instead of the standard 0.9 parasites from the mixed population of wild type and ap4ah(-) parasites. All mice became infected, but genotyping by PCR revealed that only wild type had survived (Supplementary Figure 15A). These results suggest that lack of Ap4AH causes too severe a growth defect and thus, like dUTPase, the protein is required for rodent malaria infection.
Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening
Published in mAbs, 2019
Rama Devudu Puligedda, Rashmi Sharma, Fetweh H. Al-Saleem, Diana Kouiavskaia, Arul Balaji Velu, Chandana Devi Kattala, George C. Prendergast, David R. Lynch, Konstantin Chumakov, Scott K. Dessain
We performed 2 cell fusions with PBMCs from the PV-immune subjects, P3 and P6. We fused 5 × 105 CD27+ PBMCs to LCX OCMS™ cells and plated in five 96-well plates at 1000 PBMCs/well. PV-specific mAbs were identified by whole PV ELISA.22 A total of 8 pools with OD450 2.0 or greater were selected from the P3 and P6 samples, of which 7 gave rise to stable monoclonal hybridomas following limiting dilution cloning. PV mAb neutralization titers were determined using the microneutralization test.39,40 We used the PV strains Sabin US reference stock NC2 (Type 3) and wild type Saukett (Type 3), provided by Dr. Emmanuel Vidor, Sanofi-Pasteur.