Cell lines were lifted with Accutase (Sigma-Aldrich), and 1 x 106 cells were used per sample. Monoclonal primary antibodies, By2 (anti-CD74), ab55445 (anti-MIF) and 156-3c11 (anti-CD44) were employed in indirect immunofluorescence staining. Cells were preincubated with saturating concentrations of primary antibody, followed by washing and labelling with FITC-conjugated goat anti-mouse IgG (Bio-legend). For cell surface staining, cells were fixed with 4% formaldehyde solution and washed with 1X phosphate-buffered saline (PBS). The cells were then blocked with blocking buffer (PBS / 0.1% BSA, bovine serum albumin) and washed in PBS. Primary and secondary antibodies were diluted with 0.1% BSA in PBS. Cells were sorted on a BD FAS Aria and analysed by FlowJo 8.8.6.
Fitc conjugated goat anti mouse igg
Manufactured by BioLegend
FITC-conjugated goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) antibodies. It is conjugated to the fluorescent dye fluorescein isothiocyanate (FITC), which can be detected using flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Accutase, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Syto84, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Lsrii fortessa analyser, by BD (1 mentions)
4 protocols using fitc conjugated goat anti mouse igg
1
Immunofluorescence Staining and Flow Cytometry
2
Flow Cytometry Analysis of CD74 and CD44
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Cell lines were lifted with Accutase (Sigma-Aldrich) and 1 × 106 cells were used per sample. Monoclonal antibodies By2 (anti-CD74) and 156-3c11 (anti-CD44) were employed in indirect immunofluorescence staining. Cells were preincubated with saturating concentrations of primary antibody, followed by washing and labeling with FITC-conjugated goat anti-mouse IgG (Bio-legend). For cell-surface staining, cells were fixed with 4% formaldehyde solution, and washed with 1X phosphate-buffered saline (PBS). The cells were then blocked with blocking buffer (PBS/0.1% BSA, bovine serum albumin) and washed in PBS. Primary and secondary antibodies were diluted with 0.1% BSA in PBS. Cells were sorted on a BD FACSAria and analyzed by FlowJo 8.8.6.
3
Recombinant EpCAM Protein Production
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The recombinant plasmid PGEX-4T3-EpCAM and the monoclonal antibodies of EpCAM (FMU-EpCAM-2A9 and FMU-EpCAM-2D7) are all prepared in our lab. Fetal bovine serum and mRNA isolation kit are purchased from Gibco. FITC conjugated goat anti-mouse IgG is bought from Biolegend. Mouse anti-GST antibody, protein ultrafiltration centrifugal tube, and PVDF membrane are from Millipore. The primers used were synthesized by Shanghai Sangon Biotech Company. The sequences of the primers were listed in Table 1 .
4
Flow Cytometric Analysis of Antibody Binding to Malaria-Infected Erythrocytes
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To assess binding of IgG within immune serum to pRBC in vivo, blood from immune serum (or control non-immune serum) recipient mice was diluted (RPMI, 5 U/ml heparin sulphate) and for positive controls only, incubated with an anti-mouse RBC, mouse IgG2a antibody, clone 34-3C (1.5μg/ml; Abcam, Cambridge, MA, USA). RBC were washed three times in cold FACS buffer (1% w/v BSA, 5mM EDTA in PBS) prior to incubation on ice with detection reagent, FITC-conjugated goat anti-mouse IgG (3μg/ml; Biolegend, San Diego, CA). RBC were again washed three times, prior to staining with cell-permeant RNA/DNA stain, Syto84 (5 μM; Life Technologies) and DNA stain, Hoechst 33342 (10 μg/ml; Sigma), followed by immediate flow cytometric analysis on an LSRII Fortessa analyser (BD Biosciences) and FlowJo software (Treestar, CA, USA).
To assess in vitro immune serum IgG binding to pRBC, the same protocol was employed, except that RBC were first incubated in vitro with immune serum diluted in RPMI, and then stained as above. To further investigate IgG specificity for parasites, pRBC from infected mice were first fixed and permeabilised to expose parasite structures to antibodies (BD Cytofix/Cytoperm; BD Life Sciences), and were then stained and analysed as above with the exception that reagents were prepared in Cytofix/Cytoperm Wash buffer.
To assess in vitro immune serum IgG binding to pRBC, the same protocol was employed, except that RBC were first incubated in vitro with immune serum diluted in RPMI, and then stained as above. To further investigate IgG specificity for parasites, pRBC from infected mice were first fixed and permeabilised to expose parasite structures to antibodies (BD Cytofix/Cytoperm; BD Life Sciences), and were then stained and analysed as above with the exception that reagents were prepared in Cytofix/Cytoperm Wash buffer.
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