Computational protocol: Autoregulation and Heterogeneity in Expression of Human Cripto-1

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Protocol publication

[…] Cells were detached using enzyme free dissociation buffer (Invitrogen), washed using PBS with 0.1% BSA and incubated in PBS with 2% BSA for 15 min at room temperature. Subsequently, cells were stained for cell surface CR-1 and MDR1 using mouse anti-human Cripto-1-PE (R&D system) and mouse anti-human P Glycoprotein-FITC (Abcam) respectively as per the manufacturers’ protocols. Wherever required, cells were also stained by corresponding isotype control antibodies. Single or double stained cells were analyzed in FACSCalibur flow cytometer (BD Biosciences) and data was collected using CellQuest Pro software (BD Biosciences). CR-1 and MDR1 were detected in FL2-H and FL1-H respectively, both in log mode. For each sample, data of 20,000 cells were collected. Data was analyzed using FCS Express 4 (De Novo Software). Data of each sample was gated first in FSC-SSC dot plot and then in the histogram for FL2-H to remove debris, and extreme data points. In general, more than 97% cells were retained after gating for further analysis. CR-1 positive population was identified by Overton histogram subtraction []. Cells treated with highest amount of CR1-GST and stained with isotype control antibody were used as control for subtraction.For cell sorting, U-87 MG cells were treated with CR1-GST (400 ng/ml) for 24 hr and stained with mouse anti-human Cripto-1-PE mAb or Isotype control antibody as mentioned above. CR-1 positive and CR-1 negative populations were sorted out using FACSAria III (BD Biosciences) equipped with BD FACSDiva 6.0 software. Post sorting, total RNA was isolated using TRI reagent. [...] Two-way ANOVA or one-way ANOVA was used depending upon the type of experiment. Mann-Whitney Rank Sum Test was used for data derived from real-time PCR experiments. SigmaPlot was used to generate graphs, for data fitting and statistical analysis. Means of multiple data points were plotted, as mentioned in figure legends. Error bars represent standard deviations. […]

Pipeline specifications

Software tools BD FACSDiva, SigmaPlot
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Neoplasms