Continuous xenografts from children with B-ALL or T-ALL had been previously established in immune-deficient mice as described previously.30 (link) PDXs were then harvested from spleen- and BM of engrafted mice with >95% human CD45+ by Ficoll gradient centrifugation and cryopreserved at >95% purity. For ex vivo culture experiments, PDX cells were retrieved from cryostorage and thawed at 37°C and washed twice in RPMI1640 media (Merck KGaA, Germany) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) with centrifugation at 500 × g, 5 mins. The cell density and viability were calculated using 0.4% Trypan blue (Sigma-Aldrich) assay. The PDXs were then resuspended at the appropriate cell density in QBSF-60 media (Quality Biological) supplemented with 20 ng/mL Fms-like tyrosine kinase 3 ligand (Flt-3L; ProSpec). Biological replicates were defined as the same passage of the PDX but harvested from different mice. PDX cells used in this study were obtained under approval from the University of New South Wales Animal Care and Ethics Committee (ACEC 16/168B) according to the Australian code for the care and use of animals for scientific purposes (8th Edition 2013).
Qbsf 60 media
Manufactured by Quality Biological
Sourced in United States
QBSF-60 is a cell culture media formulation designed for the growth and maintenance of a variety of cell lines. It is a complex, serum-free medium that supports the proliferation and viability of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fms like tyrosine kinase 3 ligand flt 3l, by ProSpec (2 mentions)
Trypan blue, by Merck Group (1 mentions)
Rpmi 1640 media, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Atplite 1step, by PerkinElmer (1 mentions)
G418 solution, by Merck Group (1 mentions)
4 protocols using qbsf 60 media
1
Xenograft Model for Pediatric Leukemia
2
PIM Inhibitors' Effect on T-ALL PDX Cells
3
Lentiviral Transduction of Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen viral supernatants were thawed and diluted 1:1 in serum-free QBSF-60 media (Quality Biological, Gaithersburg, MD, United States) and 8 μg/ml of protamine sulfate (Calbiochem, San Diego, CA, United States). BM-MSC, UCT-MSC, UCT-EPC, and AF-MSC were transduced at a very low multiplicity of infection (MOI) (approximately, 1 vector to 300 cells, infectious viruses per cell; MOI 0.0032) to ensure single integration events. After overnight incubation at 37°C, cells were cultured in their respective growth medium. Twenty-four hours later, cells underwent antibiotic selection using 350 μg/ml G418 solution (Sigma-Aldrich, St. Louis, MO) for UCT-EPC, or 500 μg/ml G418 disulfate salt solution for BM-MSC, UCT-MSC, and AF-MSC for 5 days, replacing the selection media after 3 days. After antibiotic selection, the remaining transduced cells expressed the neomycin resistance gene (neomycin phosphotransferase II) and the HSQ transgene. Upon confluence, transduced cells were used for supernatant collection, qPCR, and immunofluorescence imaging.
4
FVIII:C Activity Quantification in Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At about 70% confluence, all cell populations (that were plated at the same initial density) were cultured in serum-free QBSF-60 media (Quality Biological, Gaithersburg, MD, United States) for 48 h. Supernatants were collected at 48 h and the total number of cells was counted. The media was centrifuged at 1500 RPM for 5 min at 4°C to remove cellular debris, and the remaining supernatant was collected and stored frozen at −80°C until use. Aliquots of each frozen supernatant were brought to the Wake Forest Baptist Medical Center Special Hematology Laboratory, where FVIII:C activity was measured by aPTT clotting time using the facility’s clinical coagulometer, appropriately adjusting the standards to enable accurate quantitation of the low levels of FVIII present in the culture supernatants.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!