NK cells were first washed with 1x PBS (Hyclone) once and re-suspended into 1 mL AIM-V (Invitrogen) supplemented with 5% AB serum (Valley Biomedical). APC-conjugated CD107a antibody (BD Biosciences) and Golgi stop containing monensin (BD Biosciences) were added in and mixed up well with the cells. Target cells were washed and re-suspended into AIM-V (Invitrogen) supplemented with 5% AB serum (Valley Biomedical), and subsequently seeded into a U-bottom 96-well plate (Thermo Fisher Scientific) at the concentration of 2 × 105 cells/100 μL/well. NK cells were then co-cultured at a 1:1 E: T ratio with target cells in a 37°C, 5% CO2 incubator for 5 h. After incubation, the cells were used for anti-CD56 PE antibody staining (Miltenyi). The cells were then suspended in 150 μL autoMACS Running Buffer (Miltenyi) for the detection of the CD107a surface expression on NK cells by BD Accuri C6 Flow Cytometer (BD Biosciences).
Ab serum
Manufactured by Valley Biomedical
Sourced in Germany
AB serum is a type of cell culture media supplement derived from the plasma of individuals with the AB blood type. It is commonly used in cell culture applications to support the growth and maintenance of various cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Aim 5, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glass fiber filter, by PerkinElmer (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
3h thymidine, by PerkinElmer (1 mentions)
Flt3 ligand, by BioLegend (1 mentions)
Stem cell factor (scf), by BioLegend (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
X vivo 20, by Lonza (1 mentions)
N acetyl l cysteine, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by BioLegend (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Immunotools (1 mentions)
Iscove modified dulbecco media (imdm), by GE Healthcare (1 mentions)
Trypan blue exclusion assay, by Lonza (1 mentions)
Human il 15 quantikine elisa kit, by R&D Systems (1 mentions)
6 protocols using ab serum
1
NK Cell Degranulation Assay
2
Antigen-Specific T-Cell Proliferation Assay
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PBMC were thawed and suspended in RPMI 1640 medium supplemented with Minimum Essential Medium non-essential amino acids, L-glutamine, penicillin/streptomycin, sodium pyruvate, 2-mercapto-ethanol (all from Invitrogen) and heat inactivated AB serum (Valley Biomedical). This ‘complemented’ RPMI 1640 medium is hereafter termed ‘complete medium’. During the assay, PBMC were tested in triplicate and either kept in complete medium only (unstimulated control cultures) or stimulated with 2 µg/mL of antigens for 5 days at +37°C in an atmosphere of 5% CO2 in air, at which time 10 µCi [3H]-thymidine (Perkin Elmer) was added to each microculture. After 16 to 20 h at +37°C and 5% CO2, the cultures were harvested onto glass-fiber filters (Perkin-Elmer) using a multichannel cell harvester (Inotech) and incorporation of [3H]-thymidine was measured by liquid scintillation counting (MicroBeta counter Trilux from Wallac). The antigens used for lymphocyte stimulation were the vaccine antigens GI.4 Chiba 407 (1987) and GII.4 Aomori 2 (2006). Results are expressed as stimulation index (SI = mean counts per minute of antigen-stimulated cultures/mean counts per minute of control cultures).
3
Culturing 293T, T-cells, and CD34+ Cells
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293T cells were maintained in Dulbecco’s Modification of Eagle’s Medium supplemented with glutamax, non-essential amino acids, penicillin/streptomycin and 10% fetal bovine serum ThermoFisher (Waltham, MA, USA). Cells were maintained at 37 °C and 5% CO2.
T-cells were grown in X-VIVO-20 (Lonza, Allendale, NJ, USA) with 10% AB serum (Valley Biomedical, Winchester, VA, USA), 300 IU of IL-2 and 5 ng/mL each of IL-7 and IL-15 (PeproTech, Rocky Hill, NJ, USA), N-Acetyl-l -Cysteine, penicillin/streptomycin, and Gluta-MAX-I each from ThermoFisher (Waltham, MA, USA).
CD34+ hematopoietic stem cells were cultured in StemSpan SFMII media containing 1 µM SR1 each from STEMCELL Technologies, Cambridge, MA, USA and human cytokines Flt-3 ligand (100 ng/mL), SCF (100 ng/mL), TPO (100 ng/mL), IL-6 (100 ng/mL) all from Biolegend, San Diego, CA, USA.
T-cells were grown in X-VIVO-20 (Lonza, Allendale, NJ, USA) with 10% AB serum (Valley Biomedical, Winchester, VA, USA), 300 IU of IL-2 and 5 ng/mL each of IL-7 and IL-15 (PeproTech, Rocky Hill, NJ, USA), N-Acetyl-
CD34+ hematopoietic stem cells were cultured in StemSpan SFMII media containing 1 µM SR1 each from STEMCELL Technologies, Cambridge, MA, USA and human cytokines Flt-3 ligand (100 ng/mL), SCF (100 ng/mL), TPO (100 ng/mL), IL-6 (100 ng/mL) all from Biolegend, San Diego, CA, USA.
4
Expansion of EBV-specific T cells
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EBV-specific memory T cells were analyzed after stimulation with EBNA-1 or CMV-pp65 peptides and expansion in vitro as recently described [62] (link). After overnight incubation of PBMCs in IMDM (PAA Laboratories, Cölbe, Germany) containing 10% AB serum (Valley Biomedical, Winchester, VA, USA) and supplemented with Penicillin/Streptomycin 100× and L-glutamine at 2 mM (both Biochrom, Berlin, Germany) at 37°C in 5% CO2, in 96-well round bottom plates at a concentration of 2×105 cells per well with 50 IU/mL rhIL-2 (Chiron-Behring, Liederbach, Germany) and 10 ng/mL IL-7 (ImmunoTools, Friesoythe, Germany). On day 3, 5 and 7 media and IL-2 at 50 ng/µl were renewed. IL-7 at 5 ng/µl was added on day 7 of culture, and cells were harvested, washed and stained for cytokines.
5
Evaluating CAR-NK Cell Cytokine Production and Recovery
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CAR-NK cells or CAR/IL15-NK cells (4 × 106/mL) were cultured with or without HCT-116 cells (E:T = 5:1) in 24-well plates (Thermo Fisher Scientific) for either 3 h or 24 h. The supernatant was collected for measuring of IL-15 production with the Human IL-15 Quantikine ELISA Kit (R&D System, Minneapolis, MN) following the manufacturer's protocol.
To test NK cell recovery in vitro, CAR-NK cells or CAR/IL15-NK cells (1 × 106/mL) were seeded into the 24-well plates (Thermo Fisher Scientific) with AIM-V (Invitrogen) supplemented with 5% AB serum (Valley Biomedical) and cultured with low concentration (10 IU/mL) of IL-2 or without IL-2 for 7 days. The IL-2 was replenished every 2 to 3 days. The viable cell number was determined by trypan blue exclusion assay (Lonza) and the CAR percentage was analyzed by flow cytometry (BD Biosciences).
To test NK cell recovery in vitro, CAR-NK cells or CAR/IL15-NK cells (1 × 106/mL) were seeded into the 24-well plates (Thermo Fisher Scientific) with AIM-V (Invitrogen) supplemented with 5% AB serum (Valley Biomedical) and cultured with low concentration (10 IU/mL) of IL-2 or without IL-2 for 7 days. The IL-2 was replenished every 2 to 3 days. The viable cell number was determined by trypan blue exclusion assay (Lonza) and the CAR percentage was analyzed by flow cytometry (BD Biosciences).
6
KRAS Sequencing and Monocyte Differentiation
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Cell lines and normal cells were purchased from the suppliers listed in supplementary table S6 and cultured in media recommended by the supplier. KRAS (exon 2 region) was sequenced from PCR products amplified from gDNA isolated using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) at Eurofins Genomics (Ebersberg, Germany).
PBMCs were either purchased cryo-frozen from commercial suppliers (Cellular Technology Limited (CTL), OH & Tissue Solutions, Glasgow, UK) or prepared from healthy donor leukopaks (ALLCELLS, CA) by Ficoll density gradient centrifugation. CD14 + monocyte cells isolated by negative selection from PBMCs using Classical Monocyte Isolation Kit (Miltenyi, North Rhine-Westphalia, Germany) were differentiated for 4–6 days in Gibco™ AIM V Medium (Thermo Fisher Scientific, MA) with Penicillin-Streptomycin (Gibco™), 5% AB serum (Valley Biomedical, VA), 1000 U/ml GM-CSF and 500 U/ml IL-4 (Biotechne, MN) to generate iDC. Where relevant, T cells were isolated from PBMC by immunopurification using a Pan T Cell Isolation Kit (Miltenyi) according to the manufacturer’s instructions. Rabbit anti-RAS, –RASG12D and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-GAPDH was purchased from Merck Millipore (MA). Purified synthetic peptides were purchased from Peptide Protein Research Ltd, UK
PBMCs were either purchased cryo-frozen from commercial suppliers (Cellular Technology Limited (CTL), OH & Tissue Solutions, Glasgow, UK) or prepared from healthy donor leukopaks (ALLCELLS, CA) by Ficoll density gradient centrifugation. CD14 + monocyte cells isolated by negative selection from PBMCs using Classical Monocyte Isolation Kit (Miltenyi, North Rhine-Westphalia, Germany) were differentiated for 4–6 days in Gibco™ AIM V Medium (Thermo Fisher Scientific, MA) with Penicillin-Streptomycin (Gibco™), 5% AB serum (Valley Biomedical, VA), 1000 U/ml GM-CSF and 500 U/ml IL-4 (Biotechne, MN) to generate iDC. Where relevant, T cells were isolated from PBMC by immunopurification using a Pan T Cell Isolation Kit (Miltenyi) according to the manufacturer’s instructions. Rabbit anti-RAS, –RASG12D and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-GAPDH was purchased from Merck Millipore (MA). Purified synthetic peptides were purchased from Peptide Protein Research Ltd, UK
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