Peripheral blood (PB) samples were obtained from previously untreated patients fulfilling diagnostic and immunophenotypic criteria for CLL at the Leukemia Department at MD Anderson Cancer Center after informed consent and Institutional Review Board approval (Table I ). All functional assays were performed on fresh CLL B cells, negatively selected from PB samples by EasySep Human B cell enrichment kit (StemCell Technologies, Vancouver, Canada), isolated by density gradient centrifugation over Ficoll-Plaque (GE Healthcare, Little Chalfont, UK), and cultured at 107 cells/mL in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS; Gibco, Waltham, MA), and penicillin-streptomycin-L-Glutamine solution (Corning Inc., Corning, NY) (complete RPMI). Purity of all preparations was ≥ 95%, as assessed by CD19 and CD5 positivity by flow cytometry. Polyclonal goat F(ab’)2 fragments to human IgM, to human IgD or to human kappa light chain (Southern Biotechnology, Birmingham, AL) were used for cell stimulation (anti-IgM, anti-IgD, anti-Igκ), and were dialyzed against PBS prior to use in Slide-A-Lyzer Dialysis Cassettes 10.000 MWCO (Thermo Fisher Scientific). Ibrutinib was provided by Pharmacyclics, resuspended at 100 mM in DMSO and used at a final concentration of 1 μM.
Slide a lyzer dialysis cassettes 10.000 mwco
Manufactured by Thermo Fisher Scientific
The Slide-A-Lyzer Dialysis Cassettes 10,000 MWCO are a dialysis product designed for protein purification and sample preparation. The cassettes feature a semipermeable membrane with a molecular weight cutoff of 10,000 daltons, allowing for the selective removal or exchange of small molecules while retaining larger biomolecules.
Automatically generated - may contain errors
2 protocols using slide a lyzer dialysis cassettes 10.000 mwco
1
Isolation and Stimulation of CLL B Cells
2
Diphtheria Toxoid Thermal Stability Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Diphtheria
toxoid was obtained from a manufacturer within the IMI-funded VAC2VAC
consortium.26 The protein concentration
(determined by BCA (Thermo Fisher)) was adjusted to 100 μg/mL.
The toxoid was then thoroughly dialyzed (Slide-A-Lyzer Dialysis cassettes
10000 MWCO, Thermo Scientific) against a phosphate buffer (10 mM,
pH 7.2, prepared from a 1 M solution from Sigma-Aldrich). The protein
concentration was confirmed to still be approximately 100 μg/mL
by BCA. Subsequently, aliquots of the toxoid were incubated at different
temperatures. Three samples were prepared per incubation temperature.
The samples were incubated at 4, 37, 45, 50, 55, 60, and 65 °C
for 2 days.
toxoid was obtained from a manufacturer within the IMI-funded VAC2VAC
consortium.26 The protein concentration
(determined by BCA (Thermo Fisher)) was adjusted to 100 μg/mL.
The toxoid was then thoroughly dialyzed (Slide-A-Lyzer Dialysis cassettes
10000 MWCO, Thermo Scientific) against a phosphate buffer (10 mM,
pH 7.2, prepared from a 1 M solution from Sigma-Aldrich). The protein
concentration was confirmed to still be approximately 100 μg/mL
by BCA. Subsequently, aliquots of the toxoid were incubated at different
temperatures. Three samples were prepared per incubation temperature.
The samples were incubated at 4, 37, 45, 50, 55, 60, and 65 °C
for 2 days.
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!