Human NK cells were isolated from peripheral blood mononuclear cells (PBMC) (Stem Cell Technologies) by using EasySep Human NK Cell Isolation Kit (Stem Cell Technologies). In brief, 5×107 PBMCs were incubated with 50 µL Human NK Cell Isolation Cocktail in a total 2 mL PBS containing 1% FBS for 5 min at room temperature, then added to 50 µL Dextran RapidSpheres and then immediately gently mixed. After 3 min incubation in the magnet, the enriched cell suspensions were centrifuged and harvested. The isolated NK cells were expanded by using NK Cell Activation/Expansion Kit (Miltenyi Biotec). A 5×106 isolated NK cell was included in 15 mL of X-VIVO15 medium (Lonza) containing mixed biotinylated CD335 and CD2 antibodies and Anti-Biotin MACSiBead Particles (bead-to-cell as 1:2) supplemented with 500 IU/mL interleukin-2 (Peprotech) and 10% platelet-rich plasma. The medium was refreshed every 2–3 days for a total of 3 weeks’ culture. After that, the expanded cells were counted, and its purity and phenotypical markers were determined by flow cytometry using specific antibodies.
Nk cell activation expansion kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The NK Cell Activation/Expansion Kit is a laboratory equipment product designed to activate and expand natural killer (NK) cells. It provides the necessary components to stimulate the proliferation and activation of NK cells in a controlled in vitro environment.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Easysep human nk cell isolation kit, by STEMCELL (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
Human nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Facscalibur, by BD (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Automacs pro separator, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Miltenyi Biotec (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Molt 4, by RIKEN Cell Bank (1 mentions)
Jurkat, by RIKEN Cell Bank (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Recombinant human interleukin 2 il 2, by R&D Systems (1 mentions)
Nk isolation kit, by Miltenyi Biotec (1 mentions)
7 protocols using nk cell activation expansion kit
1
Isolation and Expansion of Human NK Cells
2
Isolation and Expansion of Human NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NK cells were isolated using a human NK cell isolation kit (Miltenyi Biotech) and negative selection from buffy coats of healthy donors as directed by the manufacturer. Flow cytometry assessments of CD56+ and CD3−markers (FACSCalibur Becton Dickinson, United States) were used to characterize NK cells. MiltenyiBiotech’s NK Cell Activation/Expansion Kit was used to expand the NK cells. After the first 6 days, NK colonies were detected, and then every 3 days, a new RPMI 1640 medium and 10% FBS were given to the cells for 18 days, with IL-2 used for further activation (500 IU/ml) (Miltenyi Biotech).
3
Isolation and Expansion of NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) obtained from a healthy donor were separated by a density gradient at 1500 rpm for 30 min using Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA). NK cells were separated using anti-CD56 antibody-coated magnetic micro beads in an autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany) and expanded using an NK Cell Activation/Expansion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol.
4
Murine Bone Marrow-Derived MSC Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MSCs were generated from bone marrow flushed out of tibia and femur of 4–6 weeks old BALB/c mice. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (all from Invitrogen, Carlsbad, CA) as previous report23 (link). Murine 4T1 cells were cultured in RPMI-1640 medium with 10% FBS, supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Mouse spleens were disaggregated into 10 ml RPMI-1640 medium to isolate splenocytes. Murine NK cells were prepared by NK Cell Activation/Expansion Kit (Miltenyi Biotec) and cultured in RPMI-1640 medium supplemented with 10% FBS. All cells were cultured at 37 °C in a 5% CO2 humidified atmosphere.
5
Derivation and Culture of Murine MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MSCs were derived from bone marrow flushed out of tibia and femur of 4-6 weeks old mice as described by Heng Zhu et al [33 (link)]. About 3×106 cells per mouse were obtained after passage for three times. Cells were considered as purified MSCs and identified by adipocytes and osteoblasts differentiation as described in our previous studies [34 (link)–36 (link)]. MSCs were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Invitrogen, Carlsbad, CA) as previously reported [33 (link)]. Murine RM-1 and TRAMP-C2 (PC2) cells were cultured in RPMI-1640 medium (Invitrogen) with 10% FBS, supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. Murine NK cells were prepared by NK Cell Activation/Expansion Kit (Miltenyi Biotec) and cultured in RPMI-1640 medium supplemented with 10% FBS. All cells were cultured at 37°C in a 5% CO2 humidified atmosphere.
6
Isolation and Expansion of NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MOLT-4, Jurkat, CEM and RPMI8226 cells were obtained from the RIKEN Cell Bank and maintained in RPMI-1640 (Gibco, Gaithersburg, MD) medium with 10% foetal bovine serum (FBS) (Gibco, Gaithersburg, MD) at 37 °C in 5% CO2. SKOV-3/Luc cells (Cell Biolabs, San Diego, CA), a human ovarian cancer cell line with an exogenous luciferase gene, were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Gaithersburg, MD) with 10% FBS at 37 °C in 5% CO2. The experimental protocol using healthy donors was approved by the Internal Review Board of the National Centre for Global Health and Medicine (Ref. No. NCGM-A-000268-00). The peripheral blood was isolated from healthy donors who gave written informed consent, and PBMCs were prepared, as described previously.19 (link) For preparation of NK cells, PBMCs were expanded for 7 days using an NK Cell Activation/Expansion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), and NK cells were isolated with an EasySep Human NK Cell Isolation Kit (Veritas, Tokyo, Japan). Then, NK cells were cultured for an additional 7 days in the presence of NTP-GM protein.
7
Isolation and Expansion of NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMC) were isolated from healthy donor's buffy coat or leukapheresis (OPBG Hospital, Rome, Italy) by a density-gradient technique (Ficool-Histopaque (Eurobio;France); the healthy donors had signed a written informed consent, in accordance with rules set by the Institutional Review Board of OPBG (Approval of Ethical Committee N_969/2015 prot. N_669LB). CD56 + CD3 neg NK cells, isolated with an NK isolation Kit (Miltenyi Biotec, Inc., San Diego, CA, USA), and expanded with NK Cell Activation/Expansion Kit (adapted protocol from Navarro et al. 46 ) (Miltenyi Biotec, Inc., San Diego, CA, USA) and recombinant human interleukin 2 (IL2, 500 U/ml; R&D; USA) or recombinant interleukin 15 (IL15 10 U/ml; R&D; USA). Activated NK cells were transduced in 24-well plates pre-coated with recombinant human RetroNectin (Takara-Bio. Inc; Japan) using retroviral supernatant. Enriched NK cells were cultured in GMP-compliant media (NK MACS Miltenyi Biotec, Inc., San Diego, CA, USA).
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!