CAR-T cells were cultured in (45% RPMI-1640 (Corning) 45% Click’s medium (Irvine Scientific), 10% FBS (Sigma-Aldrich), 100 unit/mL of Penicillin and 100 mg/mL of streptomycin (Thermo Scientific, 15140122) with IL-7 (10 ng/mL; PeproTech) and IL-15 (5 ng/mL; PeproTech), supplemented by 10% BenchMark™ Fetal Bovine Serum (GeminiBio, 100–106), or ImmunoCult (STEMCELL, 10981) + 50 ng/mL IL-2 (PeproTech, 200–02). When indicated CAR-T were treated with CD38 inhibitor 1uM 78c (6391, Toris). CD8+ TILs were cultured in ImmunoCult (STEMCELL, 10981) + 50 ng/mL IL-2 (PeproTech, 200–02). Jurkat and patient derived tumour cell lines 10101 and 10164 were cultured in RPMI (Corning, 10–040-CV) supplemented with 10% BenchMark™ Fetal Bovine Serum (GeminiBio, 100–106) and 1% Penicillin-streptomycin (Thermo Scientific, 15140122). Patient derived cell line 10170 were cultured in SMGM (Lonza, CC-3182). All cells were cultured at 37 °C in 5% CO2 humidity and were tested routinely for mycoplasma using MycoAlert mycoplasma detection kit (Lonza, LT07–318) and appropriate positive control (Lonza, LT-07–518).
Immunocult
Manufactured by STEMCELL
Sourced in Canada
Immunocult is a cell culture media product developed by STEMCELL Technologies. It is designed to support the growth and maintenance of various cell types, including immune cells, in vitro. The product provides the necessary nutrients and growth factors to sustain cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
X vivo 15, by Lonza (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Benchmark fetal bovine serum, by Gemini Bio (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Click s medium, by Irvine Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Metformin, by Merck Group (1 mentions)
Oligomycin, by Merck Group (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Cocl2, by Merck Group (1 mentions)
Daudi, by American Type Culture Collection (1 mentions)
Mitotempo, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
16 protocols using immunocult
1
Culturing CAR-T Cells and TILs
2
Cell Line Culturing and Compound Treatments
3
Lentiviral Transduction of TCR in CD8+ T Cells
4
Investigating MDSC-mediated T-cell suppression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh PBMCs and tumor homogenates were used immediately after isolation as described above. MDSCs were isolated from tumor homeogenates, and CD8 T cells were isolated from PBMCs using CD33 microbeads (Miltenyi) or CD8 microbeads (Miltenyi). The cells were cocultured at an MDSC:T-cell ratio of 1:4 in a U-bottom plate for 5 days in complete RPMI in the presence of 30 IU of IL-2 (proleukin) and human CD2/CD3/CD28 T-cell activator at a final concentration of 25 µL/mL (Immunocult, StemCell). For Siglec-9 blocking, a Siglec-9 blocking antibody (Clone 191240, R&D Systems) was added at a final concentration of 10 µg/mL. For sialidase treatment, MDSCs were pretreated and washed before being added to the wells as described below.
The supernatants were frozen at −80 °C, and the cells were stained for flow cytometry.
The supernatants were frozen at −80 °C, and the cells were stained for flow cytometry.
5
CAR-T Cell Cytotoxicity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CAR-T cells were cultured in Immunocult (STEMCELL Technologies; supplemented with 1% PenStrep) under the following conditions for 24 hours: without treatment, with 250 μM adenosine, or with 10 mM MNA. After pretreatment, CAR-T cells were washed with PBS and cocultured with 20,000 SK-OV-3 cells [ATCC; expanded in McCoy 5A medium (Sigma-Aldrich) supplemented with 10% FBS and 1% PenStrep] in supplemented Immunocult media in triplicate at an effector-to-target ratio of 10:1. SK-OV-3 cells and SK-OV-3 cells lysed with digitonin (0.5 mg/ml; Sigma-Aldrich) were used as negative and positive controls, respectively. Following 24 hours of coculture, supernatants were collected, and lactate dehydrogenase (LDH) was measured according to the manufacturer’s instructions (LDH Glo Cytotoxicity Assay Kit, Promega). LDH supernatant was diluted 1:50 in LDH buffer. Percent killing was measured using the following formula: % killing = corrected killing/maximum killing × 100%, where corrected killing = coculture − T cells alone, and maximum killing = positive control − negative control.
6
Targeted Knockdown of Immune Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were transfected with 250–500 pmol TIM-3 siRNA (ThermoFisher Scientific, 4392420), CEACAM1 siRNA (Integrated DNA Technologies (IDT), Coralville, IA) or nonspecific siRNA scramble control (Thermo Fisher, 12935300) (see Supplemental Table 1 for siRNA sequences) using a P3 kit for the Amaxa 4D Nucleofector (Lonza). All cells were assessed for knockdown efficiency by quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry or western blotting on days 3–4 post-transfection. Freshly isolated T cells were transfected immediately post negative selection, allowed to rest for 30 min, and activated with Immunocult (Stem Cell Technologies) according to the manufacturer’s instructions. Late-stage T cells (days 10–14 post-activation) were transfected on day 10 and assessed for knockdown efficiency on day 13 post-activation.
7
CRISPR-Cas9 transfection of human T cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to electroporation, J77 cells were washed first with PBS or HBSS, and afterwards with Opti-MEM (Gibco). Cells were then resuspended in Opti-MEM at 30 × 106 cells/mL and 400 μL (12 × 106 cells/mL) were added to each 4 mm Bio-Rad cuvette together with the corresponding plasmids (a total of 10 μg of DNA). Cells were electroporated with a Gene Pulser Xcell Electroporation System (Bio-Rad) at 240 mV and 975 μF. After electroporation, J77 cells were cultured in medium containing 5% FBS. Dead cells were discarded using Biocoll Separating Solution (Biochrom, L6115) and GFP + cells were isolated by FACS sorting.
Human CD4 + T cells isolated from healthy donor’s buffy coats and activated for 2 or 3 days were nucleofected with CRISPR-Cas9 plasmids using Amaxa Human T cell Nucleofector Kit (Lonza, VPA-1002) and Amaxa Nucleofector II (Lonza). In each cuvette, a total of 5–6 × 106 pre-activated cells (2–3 days with ImmunoCult™, STEMCELL Technologies, 10971) and 1–5 μg of DNA were placed and T-020 programme was set. Nucleofected cells were rested overnight, sorted to isolate GFP + cells and αCD3αCD28 re-activated. Pre- and post-nucleofection human CD4 + T cells were cultured in X-VIVO 15 (Lonza).
Human CD4 + T cells isolated from healthy donor’s buffy coats and activated for 2 or 3 days were nucleofected with CRISPR-Cas9 plasmids using Amaxa Human T cell Nucleofector Kit (Lonza, VPA-1002) and Amaxa Nucleofector II (Lonza). In each cuvette, a total of 5–6 × 106 pre-activated cells (2–3 days with ImmunoCult™, STEMCELL Technologies, 10971) and 1–5 μg of DNA were placed and T-020 programme was set. Nucleofected cells were rested overnight, sorted to isolate GFP + cells and αCD3αCD28 re-activated. Pre- and post-nucleofection human CD4 + T cells were cultured in X-VIVO 15 (Lonza).
8
Evaluating MALT1 Inhibitor Cytotoxicity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Jurkat human T-cell line (ATCC, clone E6.1) was exposed to a range of MALT1i concentrations and assessed for viability and inhibition of cytokine expression following cell activation. Cells were cultured in RPMI/10% FBS (Thermofisher, Waltham, MA) and maintained under a concentration of 3 x 106 cells/ml. MALT1i at different concentrations were stamped by ECHO onto 384-well plates (PerkinElmer, Waltham, MA) following which cells were plated in fresh media and incubated for 30 min before stimulation with soluble α-CD3/-CD28/-CD2 (ImmunoCult, Stemcell Technologies, Vancouver, Canada) for 24 h. Supernatants were collected and processed immediately for cytokine analysis or stored at -80°C. To assess viability of cells treated with compound, cells were lysed with CTG reagent (Promega, Madison, WI), and measured by luminometer.
9
Primary Human T Cell Isolation and Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Leukopaks from deidentified healthy donors with Institutional Review Board-approved consent forms and protocols were purchased from StemCell Technologies (200-0092). For screens, residuals from leukoreduction chambers after Trima Apheresis from deidentified healthy donors with Institutional Review Board-approved consent forms and protocols were purchased from Vitalant (formerly known as Blood Centers of the Pacific). Primary Human T cells were isolated using EasySep Human T cell isolation kit (17951) according to the manufacturer’s protocol using the EasySep magnets. The cells were seeded in appropriate culture vessels and activated with Immunocult (Stem Cell Technologies, 10971) at 12.5 μl ml−1. Cells were kept in culture at a 106 cells per ml density throughout, and cultured with IL-2 at 50 IU ml−1 (unless otherwise specified). Cells were cultured in X-Vivo-15 medium which was supplemented with 5% fetal calf serum, 50 µM 2-mercaptoethanol, and 10 mM N-acetyl-l -cysteine. Peripheral blood mononuclear cells (PBMCs) were frozen down at 5 × 107 cells per vial using Bambanker (Bulldog Bio) serum-free cell freezing medium.
10
CAR T Cell Transduction and HIV Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After transduction, CAR T cells were activated with CD3/CD28 antibodies (ImmunoCult, StemCell, Vancouver BC, Canada) for 48 h. Then, cells were spinoculated with eGFP-HIV MOI 0.1 at 1250× g for 2 h and incubated with virus overnight. Infection was analyzed as eGFP expression by flow cytometry (MACSQuant 16, Miltenyi, Bergisch Gladbach, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!