Hematopoietic clonogenic assays were performed using cells collected at day 7 after lentiviral transduction and serum-containing methylcellulose media (MethoCult) supplemented with SCF, G-CSF, GM-CSF, IL3, IL6, and EPO (StemCell Technologies) according to the manufacture's protocol. Wright staining (Sigma-Aldrich) was used to evaluate the morphology of cells within colonies on cytospins.
Wright staining
Manufactured by Merck Group
Sourced in United States, Germany
Wright staining is a laboratory technique used for the differential staining of blood cells. It is a commonly used method for the identification and classification of different types of blood cells, such as red blood cells, white blood cells, and platelets. The Wright staining process involves the use of a specific dye solution that selectively stains different cellular components, allowing for the visualization and analysis of blood cell morphology under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
G csf, by STEMCELL (2 mentions)
Stem cell factor (scf), by STEMCELL (2 mentions)
Methocult, by STEMCELL (2 mentions)
Gm csf, by STEMCELL (2 mentions)
Interleukin 3 (il 3), by STEMCELL (2 mentions)
Interleukin 6 (il 6), by STEMCELL (2 mentions)
Trypan blue, by Merck Group (1 mentions)
Vacutainer, by BD (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Propidium iodide staining, by Thermo Fisher Scientific (1 mentions)
Cell f software, by Olympus (1 mentions)
4 protocols using wright staining
1
Hematopoietic Clonogenic Assay Protocol
2
Hematopoietic Clonogenic Assay Protocol
3
Isolation and Characterization of Human Neutrophils
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Human neutrophils were isolated from heparinized venous blood of healthy donors (n = 6) with dextran (Sigma-Aldrich, St. Louis, MI, USA) sedimentation followed by centrifugation over Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA) and hypotonic lysis of erythrocytes [42 (link)]. Isolation was performed at room temperature. After isolation, the cells were resuspended in mKRPG. Viability and purity of the cells were checked with trypan blue (Sigma-Aldrich, St. Louis, MI, USA) [43 (link)] and Wright staining (Sigma-Aldrich, St. Louis, MI, USA) [44 ,45 ], respectively. Serum was isolated from venous blood of the same donors taken into serum separation blood collection tubes (BD Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA). Then, coagulation tubes were centrifuged at 1200 rpm for 15 min at room temperature and the serum was collected.
4
Apoptosis and Cell Death Assay
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Cells were seeded 80000 cells/well in 6 well - plates five hours prior to the drug treatment. Cell death assay was performed on the fourth day. Cells were incubated with propidium iodide staining (PI) purchased from Molecular Probes (Invitrogen, Darmstadt, Germany). Cells were stained 20 min [1 μg/ml]. Apoptosis analysis was conducted with Wright staining. Therefore cells were seeded and treated the same way as the cell death assay. Cells were washed with PBS and then fixed and subjected to the Wright staining according to the manufacturer's instructions (Sigma-Aldrich, Taufkirchen, Germany). After staining, cells were dried and embedded. Afterwards morphological features of apoptotic cells were observed under an Olympus x71 and images were taken with cell-F software (Olympus, Tokyo, Japan). The same equipment was used for the cell death assay.
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