γδT cells were expanded from freshly isolated peripheral blood mononuclear cells (PBMCs) or from isolated pure populations of γδT cells. Cells were obtained from healthy donors following institutional review board approval. Zoledronate-based expansions of Vγ9Vδ2+ γδT cells were performed by culturing freshly isolated PBMC in RPMI-1640 medium containing 10% Fetal calf serum, 1% Penicillin/Streptomycin, 5 μM Zoledronate (Zometa – Novartis) and 100 IU/mL IL-2 (PeproTech 200–02), which was refreshed three times weekly. After 14 d of expansion, γδT cell isolation was performed using the Miltenyi γδT cell isolation kit (130-092-892) in accordance with the manufacturer's protocol. Expansion of Vδ1+ or Vδ1–/Vδ2– γδT cells were performed as previously described.5 (link) Cells were incubated at 37°C, 5% CO2.
Penicillin streptomycin
Manufactured by Novartis
Sourced in Switzerland
Penicillin/Streptomycin is a sterile, ready-to-use solution containing penicillin and streptomycin sulfate. It is commonly used in cell culture applications as an antimicrobial agent to prevent bacterial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Interleukin 2 (il 2), by Novartis (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
γδ t cell isolation kit, by Miltenyi Biotec (1 mentions)
Zoledronate, by Novartis (1 mentions)
Penicillin, by Novartis (1 mentions)
Streptomycin, by Novartis (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Human serum, by Novartis (1 mentions)
L glutamine, by Novartis (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Human il 2, by Novartis (1 mentions)
Easysep human cd4 t cell isolation kit, by STEMCELL (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
4 protocols using penicillin streptomycin
1
Expansion and Isolation of γδT Cells
2
Culturing Human NK Cell Lines
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FLYRD18, NKIRTIL00626 (link) and Daudi cells were cultured in IMDM (Thermo Fisher Scientific), supplemented with 8% FCS (Thermo Fisher Scientific) and penicillin–streptomycin (100 IU/mL penicillin, 100 μg/mL streptomycin; Sigma-Aldrich). FLYRD18 and NKIRTIL006 cells were passaged every 2–3 days with trypsin-EDTA (Thermo Fisher Scientific). The human NK cell line KHYG-1 (DSMZ) was cultured in RPMI supplemented with 8% FBS and penicillin–streptomycin (100 IU/mL penicillin, 100 μg/mL streptomycin) containing 500 IU/mL IL-2 (Novartis). All cell lines were tested for mycoplasma using PCR-based screening and found negative.
3
HLA-DP Expression in K562 Cells
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K562 cells lacking HLA class I and class II surface expression were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Lonza, Switzerland) with 10% fetal bovine serum (Thermo Fisher Scientific, USA), 200 mM L-glutamine (Lonza), and 10,000 U/ml penicillin/streptomycin (Lonza). Cells were transduced with HLA-DP using the pLZRS vector encoding for HLA-DPA1-T2A-DPB1-IRES-ΔNGFR or HLA-DPA1-IRES-GFP and HLA-DPB1-IRES-ΔNGFR. Purity of HLA-DP–expressing cells was verified by flow cytometry. T cells isolated from healthy donors were cultured in T cell medium consisting of IMDM with 5% fetal bovine serum, 5% human serum, 200 mM L-glutamine, 10,000 U/ml penicillin/streptomycin, and IL-2 (Novartis, Switzerland) as indicated below. Human samples were deidentified prior to use and the study was approved by the Institutional Review Board of the Leiden University Medical Centre.
4
Cell Line Cultivation and PBMC Isolation
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The cell lines used in the study were previously described [69 (link),70 (link),71 (link),72 (link)]. Myla 2059 and Myla 2000 were cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA #R2405) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Cromwell, CT, USA #04-007-1A) and 1% Penicillin/Streptomycin (Sigma, #P7539). HH cells were cultured in RPMI-1640 medium supplemented with 20% FBS and 1% Penicillin/Streptomycin. SeAx, was cultured in RPMI-1640 medium supplemented with 10% human serum (HS) (Copenhagen University Hospital Blood Bank), 1% Penicillin/Streptomycin and 1000 U/mL human IL-2 (Novartis, Basel, Switzerland #004184). Cells were maintained in an incubator at 37 °C with 5% CO2 and were replenished with fresh medium every two days. Isolation of PBMC from SS patients was done by Ficoll-based density-gradient centrifugation. CD4+ T cells were isolated from PBMC using EasySep™ Human CD4+ T Cell Isolation Kit (StemCell, Vancouver, BC, Canada #17952) by following the manufacturer’s protocol. Necessary approvals were obtained from the Committee on Health Research ethics (H-16025331) prior to using SS patients’ samples and the work was performed in accordance with the Declaration of Helsinki.
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