Antibodies for flow cytometry were purchased from Cell Signaling Technologies (pY397FAK #3283, pPaxilin #2541), abcam (TET3 #ab135033, 5hmU #ab19735), Novus Biologicals (laminin #NB300–144), Active Motif (5mC #39649) or BD Biosciences (pSTAT3 #557815). Secondary antibodies used were coupled to either Alexa Fluor 647 or Alexa Fluor 488 (Invitrogen) diluted up to 1:4,000 in PBS/1% BSA staining buffer. For blocking FAK activity, GSC cell lines were treated with 10 μM FAK inhibitor (Santa Cruz #sc-203950). Intracellular staining was performed after fixation of single cell suspensions with 2% paraformaldehyde and permeabilization with ice-cold 100% methanol, blocked with PBS/1% BSA for 1h at 4°C, and stained with an antibody diluted at 1:50 in PBS/1% BSA for 30 – 45 min at room temperature. Detection of DNA modification was performed upon RNA digestion using RNase A at 40 μg/ml for 10 min at 37°C prior to blocking procedure. Stained single cell suspensions were analyzed using an AccuriC6 cytometer (BD Biosciences), followed by FlowJo 7.6.1 analysis.
Laminin nb300 144
Manufactured by Active Motif
Laminin NB300–144 is a protein isolated from the basement membrane of the human placenta. It is commonly used as a cell culture substrate to promote cell attachment and growth in various cell types.
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2 protocols using laminin nb300 144
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Antibody-based Flow Cytometry Analysis
2
Antibody-based Flow Cytometry Analysis
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Antibodies for flow cytometry were purchased from Cell Signaling Technologies (pY397FAK #3283, pPaxilin #2541), abcam (TET3 #ab135033, 5hmU #ab19735), Novus Biologicals (laminin #NB300–144), Active Motif (5mC #39649) or BD Biosciences (pSTAT3 #557815). Secondary antibodies used were coupled to either Alexa Fluor 647 or Alexa Fluor 488 (Invitrogen) diluted up to 1:4,000 in PBS/1% BSA staining buffer. For blocking FAK activity, GSC cell lines were treated with 10 μM FAK inhibitor (Santa Cruz #sc-203950). Intracellular staining was performed after fixation of single cell suspensions with 2% paraformaldehyde and permeabilization with ice-cold 100% methanol, blocked with PBS/1% BSA for 1h at 4°C, and stained with an antibody diluted at 1:50 in PBS/1% BSA for 30 – 45 min at room temperature. Detection of DNA modification was performed upon RNA digestion using RNase A at 40 μg/ml for 10 min at 37°C prior to blocking procedure. Stained single cell suspensions were analyzed using an AccuriC6 cytometer (BD Biosciences), followed by FlowJo 7.6.1 analysis.
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