Cells were treated with vehicle or ASP3026 for 48 h and then plated in methylcellulose-based medium (Methocult H4230; Stemcell Technologies) mixed in RPMI 1640 (1:5). Harvested cells were suspended in methylcellulose (v/v; 1:10) and poured into 24-well plates. The plates were incubated for 5 days at 37°C in 5% CO2. Colonies were stained using p-iodonitrotetrazolium violet and visualized using the FluorChem 8800 imaging system (Alpha Innotech, San Leandro, CA).
Fluor chem 8800 imaging system
Manufactured by Bio-Techne
Sourced in United States
The Fluor Chem 8800 Imaging System is a laboratory equipment designed for the visualization and analysis of fluorescent-labeled biomolecules. The system utilizes advanced imaging technology to capture and process digital images of fluorescent samples, enabling researchers to study a range of biological processes and molecules.
Automatically generated - may contain errors
Lab products found in correlation
M mlv reverse transcriptase, by Takara Bio (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Rt pcr kit, by Takara Bio (5 mentions)
Methocult h4230, by STEMCELL (3 mentions)
Oligo dt 12 18, by Takara Bio (3 mentions)
Mtt assay kit, by Promega (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Merck Group (1 mentions)
Coomassie blue, by Thermo Fisher Scientific (1 mentions)
Tris glycine native sample buffer, by Thermo Fisher Scientific (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
11 protocols using fluor chem 8800 imaging system
1
Evaluating Hematopoietic Progenitor Cells
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated from splenic T cells and spinal cord by using the RNeasy mini kit (Qiagen, Valencia, CA) and from spleen and cerebellum by using the Ultraspec-II RNA reagent (Biotecx laboratories, Inc, Houston, TX) following manufacturer’s protocol. To remove any contaminating genomic DNA, total RNA was digested with DNase. Semi-quantitative RT-PCR was carried out as described earlier [14 (link)–16 (link)] using a RTPCR kit from Clonetech (Mountain View, CA). Briefly, 1 μg of total RNA was reverse transcribed using oligo(dT)12–18 as primer and MMLV reverse transcriptase (Clontech) in a 20 μL reaction mixture. The resulting cDNA was appropriately-diluted, and diluted cDNA was amplified using Titanium Taq DNA polymerase and primers (Table 1 ). Amplified products were electrophoresed on a 1.8% agarose gels and visualized by ethidium bromide staining. The relative expression of each gene with respect to GAPDH was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech, San Leandro, CA).
3
Cell Viability, Apoptosis, and Colony Formation Assays
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Cell viability was measured 24, 48 and 72 h after transfection with MTT assay kit (Promega, Madison, WI). Flow cytometry analysis for apoptosis was performed using Annexin V-FITC Apoptosis Detection Kit 48 h after transfection according to the manufacturer’s protocol (Sigma–Aldrich). All experiments were performed in triplicate. The colony formation assay was performed as described previously [17 (link)]. In brief, 5 × 104 cells were mixed with methylcellulose (H4100; Stemcell Technologies, Vancouver, BC, Canada) containing RPMI-1640 + 10% FBS and poured in 35 mm plate. The colonies were allowed to grow for 7–14 days, followed by staining with p-Iodonitrotetrazolium violet and counted using Fluorchem 8800 imaging system (Alpha-Innotech, San Leandro, CA).
4
Quantifying Gene Expression in Tissues
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Total RNA was isolated from splenic T cells and spinal cord by using the RNeasy mini kit (Qiagen, Valencia, CA) and from spleen and cerebellum by using the Ultraspec-II RNA reagent (Biotecx laboratories, Inc, Houston, TX) following manufacturer’s protocol. To remove any contaminating genomic DNA, total RNA was digested with DNase. Semi-quantitative RT-PCR was carried out as described earlier [13 (link), 14 (link)] using a RT-PCR kit from Clonetech (Mountain View, CA). Briefly, 1 µg of total RNA was reverse transcribed using oligo(dT)12–18 as primer and MMLV reverse transcriptase (Clontech) in a 20 µL reaction mixture. The resulting cDNA was appropriately-diluted, and diluted cDNA was amplified using Titanium Taq DNA polymerase and following primers. Amplified products were electrophoresed on a 1.8% agarose gels and visualized by ethidium bromide staining.
The relative expression of each gene with respect to GAPDH was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech, San Leandro, CA).
The relative expression of each gene with respect to GAPDH was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech, San Leandro, CA).
5
Semi-Quantitative RT-PCR Analysis of Gene Expression
6
Inhibition of 6-HB Formation by Berberine
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A native PAGE (native polyacrylamide gel electrophoresis, N-PAGE) method was used to confirm the inhibitory activity of berberine on the 6-HB formation as previously described (Liu et al. 2005 (link)). Briefly, N36 (100 µM in PBS) was preincubated with various concentrations of berberine (0, 0.05, 0.1 mg/ml) at 37 °C for 30 min and mixed with equal molar C34 (100 µM in PBS) at 37 °C for another 30 min incubation, and then the mixtures were loaded into an 18% precast N-PAGE gels (Invitrogen). Agarose gel electrophoresis was carried out at a constant voltage of 100 V at room temperature. The gels were stained with Coomassie Brilliant Blue (CBB) and imaged using a FluorChem 8800 Imaging System (Alpha Innotech).
7
Methylcellulose Assay for Clonogenic Cells
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Methylcellulose medium was prepared by mixing 1% methylcellulose stock (Methocult H4230; StemCell Technologies) with RPMI‐1640 (1 : 4) (George et al., 2014 ; Shi et al., 2009 ). Thereafter, 3.5 mL of methylcellulose medium was added to 15‐mL tubes. Harvested cells were added in a 1 : 10 (v/v) ratio. Tubes were tightly capped, and the mix was gently inverted several times. Then, 3.5 mL of the mix was divided into 24‐well plates in triplicate. Plates were placed in a humidified incubator at 37 °C in 5% CO2 for approximately 5 days and then p‐iodonitrotetrazolium violet was added for 24 h. Colonies were visualized using the FluorChem 8800 imaging system (Alpha Innotech, San Leandro, CA, USA).
8
Quantitative RT-PCR Analysis of Gene Expression
9
Inhibition of HIV-1 gp41 6-HB Formation
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The inhibition of gp41 6-HB formation by NSPD-12m was further confirmed by N-PAGE as reported previously (Liu et al., 2005 (link)). Briefly, NSPD-12m at the indicated concentrations was incubated with the peptide N36 (100 μM in PBS) at 37°C for 30 min before the addition of the peptide C34 (100 μM in PBS). After incubation at 37°C for another 30 min, the mixture was diluted in Tris-glycine native sample buffer (Invitrogen, Carlsbad, CA) and then loaded onto 10 × 1.0-cm precast Tris-glycine gels (18%, Invitrogen) at 20 µl/well. Gel electrophoresis was carried out at constant voltage of 120 V at room temperature (RT) for 2 h in Tris-glycine native running buffer. Immediately after electrophoresis, the gel was stained by Coomassie blue (Invitrogen) and imaged by the FluorChem 8800 Imaging System (Alpha Innotech, San Leandro, CA). ADS-J1 (1 mM in PBS), a small-molecule HIV-1 entry inhibitor that blocks gp41 6-HB formation, was used as a positive control (Wang et al., 2009 (link)).
10
Methylcellulose Clonogenic Assay
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Cells were plated in a methylcellulose-based medium (Methocult H4230; Stemcell Technologies, Vancouver, BC, Canada) mixed in RPMI-1640 (1:4). Harvested cells were mixed in a 1:10 (v/v) ratio with methylcellulose media in a 15 mL conical vial. Tubes were inverted to mix the contents and poured to 6-well plates without any bubbles. The plates were then incubated at 37 °C in a 5% CO2 incubator for approximately 5 days. In order to stain the colonies, p-iodonitrotetrazolium violet was added and incubated for 24 h. Colonies were visualized using the FluorChem 8800 imaging system (Alpha Innotech, San Leandro, CA).
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