500 cells were seeded in 50 uL full DMEM in white-walled 96-well plates in technical duplicates or triplicates per independent experiment. The next day, media was changed to 50uL fresh DMEM with indicated compounds. 48 h later, 50 uL CTG reagent (Promega) was added to each well and incubated for 15 min at room temperature before luminescence measurement. Individual wells of drug-treated (non-DMSO) cells were normalized to the mean of the DMSO-treated wells within an individual experiment. Statistical analysis was performed on the mean of normalized values from individual experiments within treatment groups such that the sample size of each treatment group was equal to the number of independent experiments.
Ctg reagent
Manufactured by Promega
Sourced in Germany
The CTG reagent is a laboratory product used for the detection and measurement of biological molecules or activities. It serves as a core function in various analytical and research applications without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Opera qehs, by PerkinElmer (2 mentions)
Synergy h4 hybrid microplate reader, by Agilent Technologies (2 mentions)
Nanoglo lysis buffer, by Promega (2 mentions)
Nano glo substrate, by Promega (2 mentions)
Mouse pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Human pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Anti cd3 anti cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
Mouse il 2, by R&D Systems (1 mentions)
Human il 2, by R&D Systems (1 mentions)
Rpmi medium, by R&D Systems (1 mentions)
Penicillin streptomycin, by R&D Systems (1 mentions)
Stemspan sfem 2 medium, by STEMCELL (1 mentions)
Infinite f200 pro microplate reader, by Tecan (1 mentions)
Rhscf, by STEMCELL (1 mentions)
Facslyric flow cytometer, by BD (1 mentions)
Annexin 5 apc, by Immunotools (1 mentions)
Cisplatin, by Promega (1 mentions)
Enspire 2300 multimode plate reader, by PerkinElmer (1 mentions)
384 well plate, by PerkinElmer (1 mentions)
Immunocult, by STEMCELL (1 mentions)
16 protocols using ctg reagent
1
Cell Viability Assay in 96-well Plates
2
Activated Primary T Cell Proliferation Assay
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Assays with human primary T cells were conducted by Pharmaron. Human peripheral blood mononuclear cells for these assays were sourced commercially from Sailybio (Cat. No. SLB-HP200A), with ethical approvals and informed consent for the collection. Human primary T cells were isolated from fresh peripheral blood mononuclear cells (Sailybio) using the human Pan T Cell Isolation Kit (Miltenyi Biotec) and resuspended in RPMI medium 1640 containing 10% FBS, 1% penicillin/streptomycin, and 10 ng/mL human IL-2 (R&D Systems). Mouse primary T cells were isolated from fresh spleen cells using the mouse Pan T Cell Isolation Kit (Miltenyi Biotec) and resuspended in RPMI medium 1640 containing 10% FBS, 1% penicillin/streptomycin, and 10 ng/mL mouse IL-2 (R&D Systems). Compounds R80 and T35 were added as appropriate to various concentrations. Cells were seeded into 96-well plates and incubated at 37 °C and 5% CO2 for 1 h. Human or mouse anti-CD3/anti-CD28 Dynabeads (Thermo Fisher Scientific) (78 (link)) and 100 μM cytidine (where appropriate) were then added, and cells were incubated at 37 °C and 5% CO2 for 5 d. CTG reagent (Promega) was added to cells and incubated at room temperature for 30 min, after which luminescence was recorded using a Perkin-Elmer Envision microplate reader.
3
Expansion and Viability Assay of MSCs and HSPCs
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Patient MSCs were cultured in StemMACS+XF medium. BM CD34 + HSPCs were cultured in StemSpan SFEM II medium (Stemcell Technologies, 09655) supplemented with HSPC expansion mix, containing recombinant human (rh) stem cell factor (rhSCF, Stemcell Technologies, 78062, 50 ng/ml), rh fibroblast growth factor-1 (Thermofisher scientific, 13241-013, 10 ng/ml), rh fms-like tyrosine kinase 3/fetal liver kinase-2 ligand (Stemcell Technologies, 78137, 50 ng/ml) and rh thrombopoietin (Stemcell Technologies, 78210, 10 ng/ml). The cells were seeded at a concentration of 5 × 104 cells/ml for CD34 + HSPCs and 6 × 104 cells/ml for MSCs in 48-well plates (150 µl/well) and treated using PXS-5505 or 5-AZA as described in figures and figure legends for individual experiments. Both PXS-5505 and 5-AZA were dissolved in sterile water. At the end of treatment, 150 µl of cell suspension were mixed with 150 µl of CTG reagent (Promega, G7571) and incubated for 2 min at room temperature (RT) with shaking to ensure cell lysis. The lysates were transferred into 96-well half-area white microplates (Greiner bio-one, 675074) and luminescent signal was measured using Tecan Infinite F200 Pro Microplate Reader.
4
Cytotoxicity Assay for Cell Lines
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Cells (GP2D or LS513
cells) were plated in 96-well round bottom plates (Costar 7007 ULA
Round Bottom 96 well) in 180 μL of medium (Dulbecco’s
modified Eagle’s medium for GP2D; RPMI for LS513) containing
2% FCS (1000 cells per well) and incubated overnight at 37 °C.
The next day, 20 μL of appropriate serial compound dilutions
in medium was added, spanning a concentration range of 10 μM
down to sub nanomolar concentrations. Cells were incubated for 5 days
at 37 °C in a CO2 incubator. After addition of 20
μL of the CTG reagent (Promega) and 10 to 30 min agitation,
luminescence was measured. Data analysis (dose–response curves)
was carried out using GraphPad Prism-based software.
cells) were plated in 96-well round bottom plates (Costar 7007 ULA
Round Bottom 96 well) in 180 μL of medium (Dulbecco’s
modified Eagle’s medium for GP2D; RPMI for LS513) containing
2% FCS (1000 cells per well) and incubated overnight at 37 °C.
The next day, 20 μL of appropriate serial compound dilutions
in medium was added, spanning a concentration range of 10 μM
down to sub nanomolar concentrations. Cells were incubated for 5 days
at 37 °C in a CO2 incubator. After addition of 20
μL of the CTG reagent (Promega) and 10 to 30 min agitation,
luminescence was measured. Data analysis (dose–response curves)
was carried out using GraphPad Prism-based software.
5
Cytopathic Effect Assay for Cryptosporidium Infection
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High-content imaging HCT-8 cell infection assays with C. parvum and C. hominis were conducted following established protocols27 (link) in 384-well flat, black clear-bottom plates using an Opera QEHS (PerkinElmer) imaging system. We also developed a cytopathic effect (CPE)-based assay monitoring host cell viability using CTG reagent (Promega) that did not require staining or imaging. Further details on EC50 determination for C. parvum and C. hominis using both assays in a ten-point dose–response with threefold compound dilution will be described elsewhere (A.T.C. et al., manuscript in preparation). The EC50 of KDU731 was also determined using Nluc-expressing parasites8 (link). In brief, HCT-8 cells were infected with purified Nluc-expressing parasites (1,000 oocysts per well) and incubated with different concentrations of KDU731 for 48 h. Culture supernatant was removed from the wells and 200 μl of Nano-Glo lysis buffer containing 1:50 of Nano-Glo substrate (Promega) was added to the wells. Lysate was transferred to 96-well plates and luminescence was measured on a Synergy H4 Hybrid Microplate Reader (BioTek Instruments).
6
High Content Imaging of Cryptosporidium Infection
7
Apoptosis and Cell Viability Assays
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Apoptosis detection
One day prior to experiments, 105 cells per well were seeded in a 6-well plate or 3 × 105 cells were seeded per T-25 cell culture flask. Cells were incubated with 13 µM cisplatin (Teva GmbH, Ulm, Germany) for 48 h, further collected and washed in Annexin binding buffer (ABB: 140 mM NaCl, 2.5 mM CaCl2, 10 mM Hepes, pH 7.4). Cells were stained for 10 mins with 1:20 Annexin V-APC (ImmunoTools, Friesoythe, Germany) and 1.25 µg/mL propidium iodide in 300 µL ABB. Flow cytometric analysis was performed using a FACS Lyric flow cytometer and the software BD FACSuite V1.2.1 (Becton Dickinson GmbH, Heidelberg, Germany).
Cell Titer Glo assay
In 80 µL cell culture medium, 4000 cells per well were seeded in 96-well plate. On the next day, 13 µM cisplatin was added for 48 h and 95 µL CTG reagent (Promega, Walldorf, Germany) was added. Samples were measured with the 2300 EnSpire Multimode Plate Reader (Perkin Elmer).
8
Evaluating MALT1 Inhibitor Cytotoxicity
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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
Investigating CCR5 Inhibitor Combination Therapy in Breast Cancer
10
High-throughput Cell Viability Assay
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