Human NK cells were isolated from the peripheral blood of healthy donors using RosetteSep (Stem Cell Technologies, Vancouver, BC, Canada)—which depletes cluster of differentiation 3+ T cells and red blood cells—followed by CD56 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were cultured in α Minimal Essential Medium (Welgene, Gyeongsan, Korea) with IL-15 (30 ng/ml), IL-21 (30 ng/ml), and 10−6 M of hydrocortisone (HC; Stem Cell Technologies, Canada). To investigate the effect of PGE2 on NK cell toxicity, the cells were cultured for 48 h with either control medium or thyroid cancer cell culture supernatant at 1/4 dilution.
Il 21
Manufactured by STEMCELL
IL-21 is a recombinant human cytokine that can be used for in vitro cell culture applications. It is a member of the common gamma-chain cytokine family and plays a role in the regulation of immune cell function.
Automatically generated - may contain errors
Lab products found in correlation
α minimal essential medium, by Welgene (2 mentions)
Il 15, by STEMCELL (2 mentions)
Rosettesep, by STEMCELL (2 mentions)
Hydrocortisone, by STEMCELL (2 mentions)
Hydrocortisone hc, by STEMCELL (1 mentions)
Cd56 magnetic beads, by Miltenyi Biotec (1 mentions)
Interleukin 2 (il 2), by STEMCELL (1 mentions)
Rosettesep cocktail, by STEMCELL (1 mentions)
H3122, by American Type Culture Collection (1 mentions)
4 protocols using il 21
1
Expansion and Activation of Human NK Cells
2
Isolation and Culture of Human NK Cells
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Human umbilical cord blood cells (UCBs) were collected from healthy women with full-term pregnancies, with the written informed consent of the mothers. The study was conducted in accordance with the Declaration of Helsinki, and all experimental procedures were approved by the Institutional Review Board (IRB) (IRB No. P-01-201610-31-002). Human NK cells were collected from the cord blood (CB) of healthy individuals. Briefly, the CD3 + cell fraction (T and NKT cells) of MNCs was eliminated by Rosette Sep (Stem Cell Technologies, Vancouver, Canada). The purity of CD3- cells was verified systematically by flow cytometry based on CD3 expression and then the cells were cultured in α-MEM supplemented with 10% FBS, 10 ng/mL human IL-15, 10 ng/mL IL-21, and10–6 M hydrocortisone (Stem Cell Technologies). All cytokines used for NK cell culture were purchased from PeproTech.
3
Dual-CAR T Cell Generation Protocol
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On day 1, 1 × 106 T cells freshly purified from PBMCs were activated with CD3/CD28 T Cell Activators (STEMCELL, #10971). T cells were expanded for 3 days in XF Expansion medium (STEMCELL, #10981) with addition of 10 ng/ml IL-2 (STEMCELL, #78036.2) and 10 ng/ml IL-21 (STEMCELL, #78082). On day 4, T cells were transduced with lentiviruses at an MOI of 5, as described below. On day 7, GFP+ CAR T cells were sorted by FACS and expanded with addition of 10 ng/ml IL-2, until day 13 for cytotoxicity assays, cytokine production, or in vivo. Dual-CAR T cells directed against CD276 and FGFR4 were generated by co-transducing T cells with both CD276- and FGFR4-CARs at the MOI of 2.5 each. The idea was to maintain the same total number of surface CARs expressed on the single antigen-targeting CAR T cells originally transduced at the MOI of 5.
4
Human NK Cell Culture and Isolation
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The human NK92 cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in α-minimal essential medium (Welgene) supplemented with 12.5% fetal bovine serum (FBS), 12.5% horse serum, 0.2 mM myo inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 1% antibiotics, and 20 ng/ml IL-2 in a humidified atmosphere with 5% CO2 at 37 °C. The human lung cancer lines (H358, H460, and H3122) were obtained from ATCC and cultured in RPMI medium supplemented with 10% FBS and 1% antibiotics. Primary NK cells were obtained from human cord blood provided by a local hospital (IRB approval number P01-201610-31-002), as described previously [29] (link). Briefly, CD3 cells and red blood cells were depleted from mononuclear cells with a Rosettesep cocktail (STEMCELL technologies) and CD3-depleted cells were cultured in α-minimal essential medium (Welgene) supplemented with human IL-15 (10 ng/mL), IL-21 (10 ng/mL), and 10−6 M hydrocortisone (StemCell Technologies).
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