All subsequent experiments were conducted in the absence of a liveCdead marker

All subsequent experiments were conducted in the absence of a liveCdead marker. Cell culture-dependent cell surface expression of cMET It is known that this expression level of cellular proteins also depends on confluency and the elapsed time from seeding to analysis (Kornilova (Supplementary Table SI) and therefore display much higher cell surface cMET levels. was assessed under different culture conditions for absolute receptor expression levels of the indicated antigens and the data were set in relation to mRNA, gene count and immunoblot data. We could reproducibly quantify these receptors, omit the otherwise required normalization step and demonstrate the superiority of a 1 + 1 bispecific antibody. The same antibodies were also used to quantify the number Brassinolide of proteins in intracellular vesicles in confocal microscopy. The antibodies can be stored like regular antibodies and can be coupled with different digoxigenin-labeled fluorophores which makes them excellent tools for FACS and imaging-based experiments. for 5 min. Samples were resuspended in 200 l 1 CellFix (BD) and subjected to flow cytometric analysis (BD, FACS Canto). Data acquisition comprised of SSC-A, forward scatter (FSC)-A, FSC-W and Cy5 channel. FSC threshold for events was set between 10 000 and 12 000. Photomultiplier tube (PMT) for Cy5 channel was kept constant at 446. Overall, 10 000 events of the desired and gated populations were recorded. HTS unit settings were: 100C150 l sample, flow rate 2 l/s, mixing volume 80C100 l, mixing five times with a velocity of Mouse monoclonal to CD34.D34 reacts with CD34 molecule, a 105-120 kDa heavily O-glycosylated transmembrane glycoprotein expressed on hematopoietic progenitor cells, vascular endothelium and some tissue fibroblasts. The intracellular chain of the CD34 antigen is a target for phosphorylation by activated protein kinase C suggesting that CD34 may play a role in signal transduction. CD34 may play a role in adhesion of specific antigens to endothelium. Clone 43A1 belongs to the class II epitope. * CD34 mAb is useful for detection and saparation of hematopoietic stem cells 200 l/s and a washing step Brassinolide of 200C600 l. Data analysis was performed with FlowJo (Tree Star) and XLfit (IDBS). MESF calibration beads and MESF reference standard Mean fluorescence intensity (MFI) values were translated into MESF values by the use of Cy5 MESF Calibration Beads (Bangs Laboratories). For this purpose one drop of each bead population was added into 500 l 1 Cell Fix (BD) in Brassinolide PBS made up of 2% FCS and mixed thoroughly. The same procedure was followed for the Cy5 MESF blank control. The use of a Cy5 reference standard (Bangs Laboratories) guarantees similar flow cytometric conditions between experiments and was used to calibrate the FACS Canto prior use (unified window of analysis). For this purpose, MESF calibration beads and MESF reference standard were measured at the same PMT settings as subsequently analyzed cells. Simple cellular beads The effective fluorophore to protein ratio (F/P) was determined by the use of simple cellular? anti-human IgG beads in combination with MESF calibration beads (Bangs Laboratories). To 100 l of a 10 or 100 g/ml made up of BsAb-Dig-Cy5 solution one drop of simple cellular? anti-human IgG beads was added and incubated for 30 min on ice in the dark. Samples were then washed twice with 2 ml ice-cold PBS (2% FCS) and centrifuged at 300 for 5 min. For flow cytometric analysis (BD, FACS Canto), 500 l of ice-cold PBS (2% FCS) was added to the samples which were then analyzed in the SSC-A, FSC-A, FSC-W and Cy5 channel. In total, 10 000 events were Brassinolide recorded, exported as FCS 3.0 files and analyzed with FlowJo (Tree Star). Receptor quantitation with QuantiBRITE To evaluate phycoerythrin (PE)-labeled HER3 mAb (R&D Systems) the QuantiBRITE? PE fluorescence quantitation kit was applied. It contains lyophilized pellets of four bead populations that are conjugated with different amounts of PE molecules. The beads were resuspended in 500 l PBS (2%FCS, 1 BD Fix) and analyzed in flow cytometry. Singlets were gated in the SSC and FSC plot and the resulting PE levels used to determine the Brassinolide antibody-binding capacity (ABC) of an unknown cell population. mRNA expression profiling Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, Germany). From this material, cDNA synthesis was performed using a cDNA synthesis kit (Roche Applied Science, Germany) and the resulting double-stranded cDNA was purified with a Microarray Target Purification Kit (Roche Applied Science). Purified cDNA was then transcribed into cRNA using the Roche Microarray RNA Target Synthesis Kit (T7) (Roche Applied Science) and further.