A 350n culture bag

Manufactured by Nipro

Sourced in United States, Japan

The A-350N culture bag is a laboratory equipment product designed for cell and tissue culture applications. It serves as a container for the growth and maintenance of cell cultures. The A-350N bag provides a controlled environment for the cultivation of various cell types.

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Lab products found in correlation

Cellgro scgm serum free medium, by CellGenix (3 mentions) Interleukin 2 (il 2), by Nipro (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Variomacs, by Miltenyi Biotec (1 mentions) Anti cd158e pe z27.3.7, by Beckman Coulter (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Cellgro scgm medium, by CellGenix (1 mentions)

4 protocols using a 350n culture bag

Expansion and Characterization of Human NK Cells

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In vitro expansion of human NK cells was conducted as previously described [18 (link)]. Briefly, peripheral blood mononucleated cells (PBMCs) were isolated from healthy donors by leukapheresis and CD3⁺ T cells were depleted by VarioMACS (Miltenyi Biotech). T cell-depleted PBMCs were expanded at a seeding concentration of 2 × 10⁵ cells/ml in CellGro SCGM serum-free medium (CellGenix) with 1 % autoplasma, 1 × 10⁶ cells/ml irradiated (2,000 rad) autologous PBMCs, 10 ng/ml anti-CD3 monoclonal antibody, OKT3 (Orthoclon), and 500 IU/ml IL-2 (Proleukin) in an A-350 N culture bag (NIPRO). On day 5 of culture, NK cells were fed with fresh medium containing 500 IU/ml IL-2 and 1 % autoplasma every two days without removal of preexisting culture medium to maintain a cellular concentration at 1 ~ 2 × 10⁶ cells/ml for 14 days. The viability of expanded NK cells was determined by propidium iodide staining.
In vitro expanded NK cells were stained with primary antibodies and analyzed by a flow cytometry using anti-CD56-PE-Cy5 (B159), anti-CD16-PE (3G8), anti-CD3-FITC (UCHT1), anti-NKp30-PE (P30-15), anti-NKp44-PE (P44-8.1), anti-NKp46-PE (9E2/NKp46), anti-DNAM-1-PE (DX11), anti-CD14-FITC (M5E2), anti-CD19-PE (HIB19) (BD Biosciences), anti-NKG2D-PE (149810) (R&D systems), anti-CD158a-PE (EB6Bf), anti-CD158b-PE (GL183), and anti-CD158e-PE (Z27.3.7) (Beckman Coulter).

Lee S.J., Kang W.Y., Yoon Y., Jin J.Y., Song H.J., Her J.H., Kang S.M., Hwang Y.K., Kang K.J., Joo K.M, & Nam D.H. (2015). Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain. BMC Cancer, 15, 1011.

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Expansion and Cytotoxicity Evaluation of NK Cells

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PBMCs were isolated from random healthy donors, and NK cells were expanded as described previously under the conditions of GMP at Green Cross LabCell Corp.^{17 (link)} Briefly, CD3⁺ T-cell-depleted PBMCs were expanded at a seeding concentration of 2×10⁵ cells/ml in CellGro SCGM serum-free medium (CellGenix) with 1% auto-plasma, 1×10⁶ cells/ml irradiated (2,000 rad) autologous PBMCs, 10 ng/ml of monoclonal antibody to CD3 (OKT3; Orthoclon), and 500 IU/ml of IL2 (Proleukin) in an A-350N culture bag (NIPRO). NK cells were fed fresh medium with 500 IU/ml of IL2 every 2 days until they were harvested on day 14. After expansion, the cytotoxicity of MG4101 was evaluated by flow cytometric cytotoxicity assay against K562, SNU387 and Huh7 as described.^{17 (link)} K562 and SNU387 were obtained from the ATCC and cultured in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS) (GIBCO). Huh7 was obtained from the Korean Cell Line Bank and also cultured in RPMI-1640 medium (GIBCO) supplemented with 10% FBS (GIBCO).

Kim J.M., Cho S.Y., Rhu J., Jung M., Her J.H., Lim O., Choi G.S., Shin E.C., Hwang Y.K, & Joh J.W. (2021). Adjuvant therapy using ex vivo-expanded allogenic natural killer cells in hepatectomy patients with hepatitis B virus related solitary hepatocellular carcinoma: MG4101 study. Annals of Hepato-Biliary-Pancreatic Surgery, 25(2), 206-214.

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Ex vivo Expansion of NK Cells

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Ex vivo expansion of NK cells from healthy donor PBMCs was performed by GC LabCell (Yongin, Korea) as described previously [44 (link)]. Briefly, CD3⁺-depleted cells were seeded in CellGro SCGM medium (CellGenix, Freiburg, Germany) with 1–2% donor-plasma, γ-irradiated (2000 rad, ¹³⁷Cs source) feeder PBMCs (5 × 10⁷ cells), 500 IU/mL IL-2 (Norvartis, Basel, Switzerland), and 10 ng/mL anti-CD3 monoclonal Ab OKT3 (eBioscience, San Diego, CA, USA) in an A-350N culture bag (NIPRO, Osaka, Japan). Cultured NK cells were restimulated on day 7 of the culture by adding irradiated feeder PBMCs. OKT3 was added to culture medium to preserve activating function of irradiated PBMCs to aid NK cell expansion. Fresh medium with 500 IU/mL IL-2 was fed into NK cell cultures every two days to maintain a concentration at 0.5–2 × 10⁶ cells/mL for 3-week culture. NK cells were thawed in water bath (37 °C) and used immediately for experiments without recovery period [90 (link)]. Cell count and viability were assessed by propidium iodide (PI) staining using an automatic cell counter (Digital Bio, Seoul, Republic of Korea).

Oh E., Min B., Li Y., Lian C., Hong J., Park G.M., Yang B., Cho S.Y., Hwang Y.K, & Yun C.O. (2019). Cryopreserved Human Natural Killer Cells Exhibit Potent Antitumor Efficacy against Orthotopic Pancreatic Cancer through Efficient Tumor-Homing and Cytolytic Ability (Running Title: Cryopreserved NK Cells Exhibit Antitumor Effect). Cancers, 11(7), 966.

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NK Cell Expansion and Transplant

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For NK cell production, haploidentical parent donors underwent lymphapheresis on day -28, and CD3⁺ cell–depleted peripheral blood mononuclear cells (PBMCs) were frozen at -196°C. Peripheral blood mononuclear cells were thawed (days -12, -5, and 2) 14 days before each of the three planned infusions (days 2, 9, and 16) to allow each preparation and infusion of fresh cells. The thawed PBMCs expanded as described previously under good manufacturing practice conditions [26 (link)]. Briefly, CD3⁺ cell–depleted PBMCs were expanded at a seeding concentration of 2 × 10⁵ cells/mL in CellGro SCGM serum-free medium (CellGenix, Germany) with 1% autologous plasma, 1 × 10⁶ cells/mL irradiated (2,000 rad) autologous PBMCs, 10 ng/mL anti-CD3 monoclonal antibody (Orthoclon, Switzerland), and 500 IU/mL of interleukin-2 (IL-2; Proleukin, Switzerland) in an A-350N culture bag (NIPRO, Japan). NK cells were fed fresh medium with 500 IU/mL of IL-2 every 2 days until they were harvested after 14 days. The cytotoxicity of ex-vivo expanded donor NK cells was measured using K562, SK-N-SH, and NB-1691 cells by calcein releasing assay. For peripheral blood stem cell (PBSC) collection, haploidentical parent donors received 5–10 μg/kg of G-CSF subcutaneously once daily for four days; PBSCs were collected and transplanted without manipulation on day 0.

Choi Y.B., Son M.H., Cho H.W., Ma Y., Lee J.W., Kang E.S., Yoo K.H., Her J.H., Lim O., Jung M., Hwang Y.K., Sung K.W, & Koo H.H. (2019). Safety and immune cell kinetics after donor natural killer cell infusion following haploidentical stem cell transplantation in children with recurrent neuroblastoma. PLoS ONE, 14(12), e0225998.

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