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.
Hil 7
Manufactured by Miltenyi Biotec
Sourced in Germany
The HIL-7 is a laboratory instrument designed for cell separation and isolation. It utilizes magnetic bead technology to selectively bind and separate target cell populations from complex biological samples. The core function of the HIL-7 is to provide a versatile and efficient tool for researchers to isolate and purify specific cell types for further analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Hil 15, by Miltenyi Biotec (6 mentions)
Hil 2, by Miltenyi Biotec (6 mentions)
Hflt3 l, by Miltenyi Biotec (3 mentions)
Hil 3, by Miltenyi Biotec (2 mentions)
Insulin transferrin selenium, 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)
Phycoerythrin pe, by BD (1 mentions)
Pd 1 mih4, by BD (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Advanced dmem, by Thermo Fisher Scientific (1 mentions)
Realtime glo, by Promega (1 mentions)
Human ab serum, by MP Biomedicals (1 mentions)
Celltiter glo, by Promega (1 mentions)
T cell transact, by Miltenyi Biotec (1 mentions)
Texmacs media, by Miltenyi Biotec (1 mentions)
14 protocols using hil 7
1
Murine T Cell Activation and Expansion
2
Expansion of Human Hematopoietic Cells
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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).
3
Assessing SGN-CD70A Cytotoxicity in CTCL and T-ALL
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CTCL and T-ALL cell lines were treated with various concentrations of SGN-CD70A or PBS for 72 hours. The cell proliferation was measured by CellTiter-Glo (Promega, Leiden, Netherlands), and apoptosis was analyzed by the annexin-V/PI assay.
Primary tumor cells from PDX mice were cultured in complete media, RPMI1640 with 20% human AB serum (MP, Solon, OH). For the proliferation assays, primary tumor cells from PDXs were stimulated to grow with CD2/CD3/CD28 activation beads and incubated with hIL-2 (500 U/mL) and hIL-7 (1000 U/mL) (Miltenyi Biotec) at an optimal cell density of 5 to 10 × 105 cells/mL. After 24 or 48 hours of activation, the cells were treated with various concentrations of SGN-CD70A as indicated. The proliferation of primary tumor cells was determined by Real-Time Glo (Promega) at 24, 48, and 72 hours after drug treatment. Apoptosis assays using primary tumor cells were performed as described previously.20 (link) Briefly, primary tumor cells were harvested from the spleens of PDX mice and cultured in a complete medium with 250 U/mL of hIL-2 without activation beads. At 24 hours, cells were treated with various concentrations of SGN-CD70A or ADC-IgG control. After 72 hours of treatment, cells were analyzed by the annexin-V/PI assay.
Primary tumor cells from PDX mice were cultured in complete media, RPMI1640 with 20% human AB serum (MP, Solon, OH). For the proliferation assays, primary tumor cells from PDXs were stimulated to grow with CD2/CD3/CD28 activation beads and incubated with hIL-2 (500 U/mL) and hIL-7 (1000 U/mL) (Miltenyi Biotec) at an optimal cell density of 5 to 10 × 105 cells/mL. After 24 or 48 hours of activation, the cells were treated with various concentrations of SGN-CD70A as indicated. The proliferation of primary tumor cells was determined by Real-Time Glo (Promega) at 24, 48, and 72 hours after drug treatment. Apoptosis assays using primary tumor cells were performed as described previously.20 (link) Briefly, primary tumor cells were harvested from the spleens of PDX mice and cultured in a complete medium with 250 U/mL of hIL-2 without activation beads. At 24 hours, cells were treated with various concentrations of SGN-CD70A or ADC-IgG control. After 72 hours of treatment, cells were analyzed by the annexin-V/PI assay.
4
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.
5
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.
6
Directed Differentiation of CD34+ UCB Cells to T Cells
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CD34+ UCB cells were stimulated for 16 h before transduction in the CellGenix GMP SCGM Stem Cell Growth Medium (CellGenix, 20802–500) supplemented with cytokines: 20 ng/ml human thrombopoietin, 100 ng/ml human stem cell factor, and 100 ng/ml hFLT3-L (Miltenyi Biotec). Stimulated CD34+ UCB cells were transduced with HOXA9-GFP or control GFP VSV-G lentiviral vectors. 48 h after transduction, GFP-expressing cells were sorted using FACS ARIA III. CD34+ UCB cells were also electroporated with Cas9-gRNA ribonucleoprotein complexes for HOXA9 knock-out and HOXA5-9 deletion. gRNAs were synthesized from Integrated DNA Technologies as Alt-R CRISPR-Cas9 crRNA. The functional gRNA was created after annealing with Alt-R tracrRNA (Integrated DNA Technologies). Editing efficiency evaluated with TIDE algorithm (https://tide.nki.nl/ ) was around 40%. Next, the cells were cultured on OP9-DL1 stroma cells in homemade MEMα (Thermo Fisher Scientific, 12000063) supplemented with 20% FBS HycloneSH30070.03HI (Thermo Fisher Scientific, 10772634) and cytokines: 5 ng/ml rFLT3-L, 10 ng/ml human stem cell factor, and 2 ng/ml hIL7 (Miltenyi Biotec; Six et al., 2011 (link)). Stromal OP9-DL1 cells were changed every week. At different time points of culture, cells were collected and analyzed by FACS for surface expression of TCRγδ and TCRαβ, and TCRA rearrangements by multiplex RT-PCR analysis.
7
CAR T Cell Cytotoxicity Assay for B-ALL
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Target cells (cell lines or primary B-ALL cells; 100,000 target cells/well of a 96-well plate) were incubated with CD22-CAR or mock-IC T cells at different effector:target (E:T) ratios for the indicated time periods. Cell lines were cultured in RPMI-1640, 10% FBS and penicillin–streptomycin. Primary cells were cultured in StemSpanTM SFEM media (StemCell Technologies, Vancouver, Canada), 20% FBS, penicillin–streptomycin, insulin–transferrin–selenium (Gibco/Invitrogen), hSCF (100 ng/mL), hFLT3L (100 ng/mL), hIL3 (10 ng/mL) and hIL7 (10 ng/mL, all from Miltenyi Biotec, Bergisch Gladbach, Germany). CAR T cell-mediated cytotoxicity was determined by analyzing the residual live (7-aminoactinomycin D–; 7-AAD–) target cells at each time point and E:T ratio. For absolute cell counting, BD TruCountTM absolute count tubes (BD Biosciences) were used. Quantification of the proinflammatory cytokines IL2, TNFα and IFNγ was measured by ELISA using BD OptEIATM Human ELISA kits (BD Biosciences), in supernatants harvested at 24 hours (cell lines) and 48 hours (primary cells) post T cell-exposure, using an 1:1 E:T ratio. Online supplementary table 1 shows clinic-biological features of the B-ALL samples used.
8
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.
9
Cytotoxicity Evaluation of ADCs in T-cell Malignancies
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Human CTCL cell lines (HH, H9, MJ, Hut 78) and T-cell acute lymphoblastic leukemia (T-ALL) cell lines (HPB-ALL, PF-382, CCRF-CEM, and Jurkat) were acquired from American Type Culture Collection (ATCC) and cultured in complete media recommended by ATCC. All cell lines were passaged 3 times per week and maintained at a cell density below 106 cells/mL, and logarithmically growing cells were used for all experiments.
SGN-CD70A (h1F6239C-PBD) and ADC-IgG control (hIgG239C-PBD) were provided by Seagen Inc. (Seattle, WA).17 (link) T-cell activation/expansion kit and recombinant cytokines, human IL-2 (hIL-2) and hIL-7, were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).
SGN-CD70A (h1F6239C-PBD) and ADC-IgG control (hIgG239C-PBD) were provided by Seagen Inc. (Seattle, WA).17 (link) T-cell activation/expansion kit and recombinant cytokines, human IL-2 (hIL-2) and hIL-7, were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).
10
Antigen-specific T Cell Expansion Protocol
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Splenocytes were stimulated with various doses of soluble SIINFEKL peptide (0.1 to 1000 nM) and incubated at 37°C in 10% FCS, 5 mg/mL penicillin, 5 mg/mL streptomycin, 0.05 mM 2‐ME, 10 mM HEPES, and 1 mM sodium pyruvate (all Invitrogen, CA, USA) in RPMI (cRPMI). Cells were split on day 4, following peptide stimulation and 20 U/mL of hIL‐2 (Roche) was added to each well. Long‐term cultures were split every other day, starting from day 2, into cRPMI with 10 U/mL of hIL‐2, 10 ng/mL of hIL‐7 (Miltenyi Biotec, Bergisch Gladbach, Germany) and 10 ng/mL of hIL‐15 (Peprotech, Rehovot, Israel). Unstimulated cells were cultured in the same conditions without peptide and were used as controls.
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