HEK 293 and P815 target cells were maintained in RPMI or Dulbecco modified Eagle medium, respectively (Gibco, Life Technologies, Grand Island, NY), supplemented with 10% FBS. Primary human CD8+ T cells were negatively selected from PHA blasts by using the Dynabeads Untouched Human CD8+ kit (Life Technologies), according to the manufacturer's instructions, and stimulated with irradiated (3000 rads) PBMCs and 1 μg/mL PHA (Oxoid, Hampshire, United Kingdom). Cells were grown in RPMI supplemented with 5% human serum, 100 U/mL recombinant IL-2, 1 mmol/L sodium pyruvate, 2 mmol/L l -glutamine, 0.075% sodium carbonate, and 50 μmol/L 2-mercaptoethanol to obtain CTLs. Polyclonal NK cell populations were also obtained after NK cell purification (RosetteSep Method; STEMCELL Technologies, Vancouver, British Columbia, Canada) and cultured, as previously described.11 (link)
Rosettesep method
Manufactured by STEMCELL
Sourced in United Kingdom
The RosetteSep Method is a cell separation technique used to isolate specific cell types from a heterogeneous cell population. It utilizes a cocktail of antibodies that recognize and bind to unwanted cells, forming rosettes that can be easily separated from the desired cell population.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Phytohemagglutinin (pha), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Anti igg1 pe, by Southern Biotech (1 mentions)
Lympholyte h density gradient centrifugation, by Cedarlane (1 mentions)
2 protocols using rosettesep method
1
Expansion of Primary Human CTLs and NK Cells
2
Isolation and Characterization of Polyclonal NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells were isolated by Lympholyte‐H density gradient centrifugation (Cedarlane, Burlington, Canada) from buffy coats of healthy donors, provided by the blood transfusion center of IRCCS Ospedale Policlinico San Martino (Genoa, Italy), following approved internal operational procedures (IOH78). The donor KIR genotypes were analyzed as previously described.52 NK cells were purified using the RosetteSep method (StemCell Technologies, Vancouver, BC) and cultured on irradiated feeder cells in the presence of 2 μg/ml phytohemagglutinin (Sigma‐Aldrich, Irvine, UK) and 600 IU/ml rIL‐2 (Proleukin, Chiron Corp., Emeryville, CA) to obtain activated polyclonal NK cells. NK cells were stained first with HP‐DM1 mAb followed by anti‐IgG1‐PE (Southern Biotechnology, Birmingham, AL), washed twice, and incubated with EB6B‐APC (anti‐KIR2DL1/S1 and anti‐KIR2DL3 allotypes carrying E35 and R50, Beckman Coulter, Brea, CA). 143211‐PE mAb (anti‐KIR2DL1/S5, R&D Systems, Minneapolis, MN) in combination with EB6B‐APC was also used. Flow cytometric analysis was performed on Gallios flow‐cytometer (Beckman Coulter), and data were analyzed using FlowJo Version 10.7 (BD Biosciences, San José, CA).
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!