Advanced DMEM, IMDM, RPMI-1640, l -glutamine, penicillin/streptomycin (P/S), and insulin-transferrin-selenium (ITS) were purchased from GIBCO/Invitrogen (Waltham, MA, USA). StemSpan serum-free expansion medium (SFEM) was purchased from STEMCELL Technologies (Vancouver, Canada). Phosphate-buffered saline was purchased from Merck Life Science SL (Darmstadt, Germany). Human (h) stem cell factor (SCF), hFMS-like tyrosine kinase 3 ligand (FLT3-L), hIL-3, hIL-7, and hIL-15 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Fetal bovine serum (FBS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD3 (OKT3) and anti-CD28 (CD28.2) mAbs, 7-amino-actinomycin D (7-AAD), and fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, peridin chlorophyll (PerCP)-, allophycocyanin (APC)-, phycoerythrin/cyanine7 (PE/Cy7)-, Brilliant Violet 421 (BV421)-, and BV510-conjugated mAbs specific for human CD3 (SK7), CD19 (HIB19), CD22 (HIB22), CD10 (HI10a), CD13 (WM15), CD45 (HI30), HLA-ABC (G46-2.6), CD25 (M-A251), CCR7 (150503), CD27 (L128), CD45RO (UCHL1), LAG3 (T47-530), TIM3 (7D3), PD-1 (MIH4), and isotype-matched negative control mAbs, were purchased from BD Biosciences (Franklin Lakes, NJ, USA), CD69 (REA824) from Miltenyi Biotec, and anti-His (J095G46) from BioLegend (San Diego, CA, USA).
Hil 15
Manufactured by Miltenyi Biotec
Sourced in Germany
The HIL-15 is a laboratory equipment designed for high-intensity light-based cell culture applications. It provides a controlled and consistent light environment for cell growth and development.
Automatically generated - may contain errors
Lab products found in correlation
Hil 7, by Miltenyi Biotec (6 mentions)
Hil 2, by Miltenyi Biotec (4 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Allophycocyanin, by BD (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Stemspan serum free expansion medium, by STEMCELL (1 mentions)
Hil 3, by Miltenyi Biotec (1 mentions)
Phycoerythrin pe, by BD (1 mentions)
Pd 1 mih4, by BD (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Advanced dmem, by Thermo Fisher Scientific (1 mentions)
T cell transact, by Miltenyi Biotec (1 mentions)
Texmacs media, by Miltenyi Biotec (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Microbead, by Miltenyi Biotec (1 mentions)
96 well round bottom plate, by BD (1 mentions)
2 3 cgamp, by InvivoGen (1 mentions)
8 protocols using hil 15
1
Expansion of Human Hematopoietic Cells
2
Generation of CD19-Specific CAR T Cells
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Second generation CD19-targeting CAR construct consists of an extracellular antigen-binding domain (CD19-scFV), CD8 for hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ chain signaling domain followed by EGFRt as a tag. PBMC were collected from buffy coat from healthy donors. T cells were purified on LS columns (Miltenyi Biotec, # 130-042-401) using CD4 and CD8 microbeads (Miltenyi Biotec, #130-045-101 and #130-045-201), were activated with CD3/CD28 beads (T Cell TransAct; Miltenyi Biotec, #130-111-160) and incubated for 24 h. Then, activated T cells were transduced with the lentiviral vector (pCDH-EF1a-CD19 (FMC63)-2nd(4-1BB)-EGFRt; Creative biolabs) carrying the CAR construct. Activated T cells were cultured in TexMACS media (Miltenyi Biotec, #130-097-196) with hIL-7 (155U/mL, Miltenyi Biotec, # 130-095-362) and hIL-15 (290U/mL, Miltenyi Biotec, #130-095-762). CAR positivity was confirmed by expression of EGFRt in CD45+CD3+CD19− population by flow cytometry. Expanded CART19 cells were frozen and stored in vials in liquid nitrogen before use.
3
Activation of Naive CD4+ T Cells
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Naive CD4+ T cells were plated at 0.5 × 106 cells/well (200 μl/well) in a 96-well round bottom plate and cultured in medium containing 10 ng/ml hIL-2, hIL-7 and hIL-15 (Miltenyi). Additionally, monoclonal antibodies against CD3 (0.1 μg/ml, clone CLB-T3/4.E, Sanquin) and CD28 (0.2 μg/ml, clone CLB-CD28/1, Sanquin) were added to generate activated CD4+ T cells. The supernatant was collected after 48 h of culture and frozen at −20 °C until analysis. A Human IFN-Beta ELISA Kit with high sensitivity (PBL Assay Science) was used according to the manufacturer’s instructions.
4
Activation of Naive CD4+ T Cells
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Naive CD4+ T cells were flow sorted on the basis of the CD3+HLA-DR-CD4+CD25-/lowCD45RA+ phenotype and cultured for 48-72 h after plating at 0.5 × 106 cells/well in 96-well round bottom plates (BD Falcon) in RPMI-1640 medium supplemented with 10% FBS and Antibiotic–Antimycotic Solution (Sigma) in the presence of hIL-2, hIL-7 and hIL-15 (Miltenyi, each 10 ng/ml); additionally, monoclonal antibodies against CD3 and CD28 were added to activate CD4+ T cells. In addition, CD4+ T cells were treated with a STING inhibitor (H-151, 15 ng/ml; InvivoGen) or a STING agonist (2′3′-cGAMP, 15 μg/ml; InvivoGen) for the last 8 h of culture. Intracellular IFNβ and phosphoprotein detection was performed using flow cytometry.
5
Isolation and Expansion of hTERT-Specific T Cells
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hTERT865-873 -specific TCR sequences were isolated as previously described in [38 (link)]. As control, T cells engineered with a transgenic TCR specific for epitope hHCV1406-1415 (KLVALGINAV) were used. hTERT865-873 - and hHCV1406-1415- specific T cells were obtained by transduction of OKT-3-activated PBMCs with the viral supernatant of hTERT865-873/PG13 or HCV1406-1415/PG13 cell lines in the presence of hIL-15 (hIL-15, 100 ug/ml, Miltenyi) and rIL-2 (rIL-2, 300 IU/ml; Peprotech). Selected T cells were then expanded in AIM-V medium (Gibco) supplemented with 5% human serum (Gibco) with OKT-3 (30 ng/ml; eBioscience), recombinant IL-2 and human IL-15. The percentage of CD4+ and CD8+ T cells in the culture was always tested before in vitro or in vivo studies. In general, CD8+ T lymphocytes represent about 70-80% of the total T cells and numbers for in vivo treatments were adjusted in order to inject 2.5×106 CD8+ T cells.
6
Murine T Cell Activation and Expansion
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Murine T cells were activated for 24 h with (i) αCD3/CD28 Ab-coated beads (Gibco, Thermo Fisher Scientific) at a bead to cell ratio of 2:1 and 50 IU/ml of hIL-2 (Glaxo), (ii) plate-coated αCD3 Ab (5 µg/ml; eBioscience) plus soluble αCD28 Ab (2 µg/ml; eBioscience) and hIL-2 (50 IU/ml), or (iii) Concanavalin A (2 µg/ml; Sigma), hIL-7 (1 ng/ml; Miltenyi) and 50 IU/ml hIL-2 before transduction. Transduced T cells were cultured at a concentration of 0.5–106 cells/ml in T cell medium enriched with 50 IU/ml hIL-2 only or with 10 ng/ml of both hIL-7 and hIL-15 (Miltenyi) from day 2 after transduction onwards. T cells were typically counted every 2–3 d. T cell expansion was calculated by dividing the absolute number of expanded T cells at each time point during culture by the respective number on day 0 (T cell transduction). T cell viability was assessed by trypan blue staining, and the phenotype was assessed by cell-surface staining of CD44 and CD62L followed by flow cytometric analysis.
7
Expansion and Characterization of Mouse NK Cells
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NK cells were purified from mouse spleens using Mouse NK cell isolation Kit according to manufacturer’s specifications (STEMCELL #19855). NK cells were then expanded for 6 days with hIL-15 50 ng/mL (Miltenyi #130-095-765) in RPMI 20% FCS supplemented with L-glutamine (2 mM; Gibco), penicillin/streptomycin (100 µg/mL; Gibco), Sodium Pyruvate (1 mM; Gibco) and finally FACS sorted (NK1.1+, NKp46+, CD3-, CD19-) (BD Biosciences FACSAria III). After overnight starvation in the absence of IL-15, Cish+/+Ncr1 iCre or Cishfl/flNcr1iCre NK cells were stimulated or not during 4 hours with IL-2 (Proleukin, Novartis pharma SAS) or IL-15.
8
Activated CD4+ T Cell Generation
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To generate activated CD4+ T cells, naive CD4+ T cells were first activated with monoclonal antibodies against CD3 (0.1 μg/ml, clone CLB-T3/4). E, Sanquin) and CD28 (0.2 μg/ml, clone CLB-CD28/1, Sanquin) for 48–72 h. Activated and nonactivated CD4+ T cells were cultured in the presence of 10 ng/ml hIL-2, hIL-7 and hIL-15 (Miltenyi). Ex vivo-generated cDC1s were purified by flow cytometry and were then cocultured with activated CD4+ T cells for 12 h. An anti-IFNAR2 monoclonal blocking antibody (clone MMHAR-3, 5 μg/ml; PBL Assay Science), an anti-IFNGR1 blocking antibody (5 μg/ml; R&D Systems), or mouse IgG2A isotype control (5 μg/ml; InvivoGen) were added accordingly. cDC1s cultured ex vivo without CD4+ T cells were used as controls.
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