To establish the CTC cultures, a parallel sample of PB from each patient was processed by both Ficoll gradient separation and negative immunomagnetic enrichment, as described above. Purified CTCs were incubated under normoxic conditions (5% CO2) in 48-well plates at 37 °C in Dulbecco’s modified Eagle’s medium/F12 (Gibco) containing insulin (20 ng/mL) (Gibco), 1% N2 complement (Gibco), epithelial growth factor (EGF: 20 ng/mL) (Gibco), 1% penicillin–streptomycin solution (Gibco), fibroblast growth factor-2 (FGF2: 10 ng/mL, Gibco) and 10% foetal bovine serum (FBS) (Gibco). The cultured cells were then fixed in 2% of paraformaldehyde (Sigma Aldrich), permeabilized by 0.1% Triton X-100 (Sigma Aldrich) and incubated overnight at 4 °C with a rabbit anti-Mela-A/MART1 MoAb (ab210546, Abcam) at 1:200 dilution and mouse anti-CD45 at 1:100 dilution (HI30, Novus Biologicals). Cells were then incubated at room temperature with an anti-rabbit FITC secondary MoAb (AP307F, Sigma-Aldrich), an anti-mouse Alexa Fluor 647 (A28181, Invitrogen), while TRITC-conjugated Phalloidin (P1951, Sigma Aldrich) and DAPI were used to visualize the β-actin and nucleus, respectively. Samples were analysed under a confocal laser scanning microscope (C2plus, Nikon Instr.) equipped with a dedicated software (NIS element software, Nikon Instruments).
Ap307f
Manufactured by Merck Group
Sourced in United Kingdom
The AP307F is a laboratory equipment product offered by Merck Group. It is designed to perform specific functions within a laboratory setting. The core function of this equipment is to provide reliable and consistent performance for laboratory applications. However, a detailed description of the product's features and intended use cannot be provided while maintaining an unbiased and factual approach. Therefore, the full description is not available.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Nis element software, by Nikon (1 mentions)
Ab210546, by Abcam (1 mentions)
Dulbecco s modified eagle s medium f12, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
N 2 complement, by Thermo Fisher Scientific (1 mentions)
A28181, by Thermo Fisher Scientific (1 mentions)
P1951, by Merck Group (1 mentions)
A5228, by Merck Group (1 mentions)
T9284, by Merck Group (1 mentions)
Dmrb fluorescent microscope, by Leica (1 mentions)
2 protocols using ap307f
1
Establishing Circulating Tumor Cell Cultures
2
Immunocytochemistry of Smooth Muscle Cells
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Immunocytochemistry was carried out once the primary culture was confluent after ~3 days. The SMCs were washed with ice cold PBS and then fixed with 4% PFA at RT for 30 min. The SMCs were then permeabilised using 0.15% Triton (T9284, Sigma-Aldrich, Poole, UK) at RT for a further 30 min followed by blocking with 5% milk in PBS for 1 h. The SMCs were incubated in a humidifier at 4°C overnight with primary mouse monoclonal anti-actin α-smooth muscle antibody (A5228, Sigma-Aldrich, Dorset, UK) (1:200) and rabbit anti-CBS (1:200) (H00000875-D01P). The SMCs were washed 3x with PBS and incubated with TRITC goat anti-rabbit secondary antibody (1:200) (A16101, Thermo Fisher Scientific, Waltham, MA U.S.A) and goat anti-rabbit FITC conjugated secondary antibody (1:200) (AP307F, Sigma-Aldrich, Dorset, UK) at RT for 45 min. The SMCs were then washed once with PBS then nuclear stain with DAPI (1µg/ml) for 5 min and then washed 3x with PBS followed by distilled water once. The SMCs were imaged using Leica DMRB fluorescent microscope.
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