Cell-intrinsic resistance towards momelotinib and ruxolitinib (Selleck Chemicals, Kirby Drive, Houston, USA) was evaluated as described previously [52 (link)]. Briefly, leukemic cells were exposed to a concentration range (24μM to 750 nM) of these compounds for four days and cytotoxicity was quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). For TSLP stimulation, cells were pre-incubated for 1 hour with 25 ng/ml TSLP (R&D systems, Oxon, UK). In addition to single cell cultures, leukemic cells (1*106 cells) were co-cultured with primary MSCs (5*104 cells) for four days in a 24 well plate in the presence of a dilution series of momelotinib and ruxolitinib. Cell survival was quantified using flow cytometry (MACSQuant, FlowJo 10.0.8r1), and cells were stained with Brilliant Violet 421 anti-human CD19 antibody (Biolegend), FITC Annexin V (Biolegend), and Propidium Iodide (PI; Invitrogen, Bleiswijk, Netherlands), as described previously [45 (link)].
Macsquant
Manufactured by FlowJo
Sourced in Netherlands
The MACSQuant is a compact and versatile flow cytometer designed for cell analysis and sorting. It offers precise measurements of multiple parameters for a wide range of cell types, including fluorescently labeled cells. The MACSQuant provides reliable and reproducible results, making it a useful tool for various applications in life science research and clinical settings.
Automatically generated - may contain errors
2 protocols using macsquant
1
Evaluating Leukemia Cell Resistance to Momelotinib and Ruxolitinib
2
Isolation of Skin Cells from Ear Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ear skin samples were digested in 1 mL of Dulbecco modified Eagle medium containing 20 mmol/L HEPES, 0.025 mg/mL Liberase, 396 U/mL DNase I, and 0.5 mg/mL hyaluronidase at 378C at 1400 rpm for 1 hour to isolate skin cells. Samples were passed through a 40-mm sieve, and the cell suspension was washed twice with PBS. For flow cytometry, skin or LN cells were resuspended in PBS/2% BSA. Cells (2-5 3 10 5 ) were stained with a mAb in 100 mL of staining solution for 30 minutes at 48C. Cell suspensions were washed twice and resuspended in 200 mL of PBS/2% BSA. For fluorescence-activated cell sorting analysis of intracellular cytokines, cells were incubated with Brefeldin A in RPMI 1640 with 10% FCS and 1% penicillin/streptomycin before fixation in forkhead box protein 3 Staining Buffer (eBioscience, San Diego, Calif) and staining. Analysis was done with the Miltenyi MacsQuant flow cytometer with MacsQuant or FlowJo analysis software (FlowJo, Ashland, Ore).
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!