Highlights► Microfluidic arrays enable analysis of 96 qPCR assays on 1440 single cells. ► Detailed methods on obtaining qPCR data and performing preliminary data processing. ► Data from sufficient cells to address noise inherent in single-cell transcription. ► Methods used for conventional qPCR do not necessarily apply to single-cell qPCR.

The valency of quantum dot nanoparticles conjugated with biomolecules is closely related to their performance in cell tagging, tracking, and imaging experiments. Commercially available streptavidin conjugates (SAv QDs) are the most commonly used tool for preparing QD−biomolecule conjugates. The fluorescence quenching of biotin-4-fluorscein (B4F) provides a straightforward assay to quantify the number of biotin binding sites per SAv QD. The utility of this method was demonstrated by quantitatively characterizing the biotin binding capacity of commercially available amphiphilic poly(acrylic acid) Qdot ITK SAv conjugates and poly(ethylene glycol) modified Qdot PEG SAv conjugates with emission wavelengths of 525, 545, 565, 585, 605, 625, 655, 705, and 800 nm. Results showed that 5- to 30-fold more biotin binding sites are available on ITK SAv QDs compared to PEG SAv QDs of the same color with no systematic variation of biotin binding capacity with size.

Encapsulation is known to deteriorate the performance of subcutaneous (SQ) continuous glucose monitors (CGMs), preventing these devices from meeting the long-term functionality requirements for widespread use and creating a bottleneck in artificial pancreas (AP) development.1,2 While recent studies of implanted SQ sensors have shown promising results, there is still much room for improvement, including the reduction of encapsulation-induced sensor lag. 3,4 We present a proof-of-concept study of a novel flushing assembly to routinely clean the sensor surface, thereby prolonging its lifetime. Placing the sensor in the intraperitoneal (IP) space allows flushing with saline that would not be possible in the restricted SQ space.Fluorescent glucose sensors were implanted in the SQ or IP space of sheep. Sensors were provided by the manufacturer in a lengthened, tethered format. The IP sensors were modified with silicone tubing, flush port, Dacron cuff, and adaptors to allow flushing with saline solution. Experiments were conducted under an IACUC-approved protocol by BioSurg, Inc (Davis, CA). After preliminary testing to optimize the flushing procedure, long-term responsiveness was evaluated with an IP sensor placed in 1 sheep and an SQ sensor placed in a second sheep. The IP sensor was flushed weekly with saline. Glucose response challenges were performed periodically over 3 months by infusing 0.5 g/kg dextrose through an ear vein over 60 s (13 challenges over 114 days for IP, 9 challenges over 91 days for SQ). The results are summarized in Figure 1.The IP sensor demonstrated anomalously slow response during the first challenge (day 8) due to tissue trauma following implantation, which is known to cause inflammatory response.3 Excluding day 8, the IP sensor maintained consistent responsiveness throughout the 114-day period, with time to half-maximum (t 1/2) between 2.7 and 4.7 min and time to maximum (t max ) between 11.6 and 17.2 min. Conversely, the nonflushed sensor in the SQ space gradually lost responsiveness, with t 1/2 between 2.6 and 13.5 min and t max between 9.7 and 72 min. By 91 days following implantation, the SQ sensor signal did not peak within the 60-min testing period (see Figure 1B).The development of long-term implantable CGMs is a key step toward making this technology more practical; however, CGM performance is hindered by diffusion lag and loss of sensitivity caused by encapsulation driven by the foreign body response.2,3 The IP space has already been shown to be valuable to AP applications, with experimental evidence showing both faster insulin action and faster glucose sensing in this space. 5,6 The performance of the flushed IP sensor presented here far exceeded that of the conventional SQ sensor after long implantation periods, showing promise for further investigation of the flushing method.This proof-of-concept study introduces the use of a flushing mechanism to allow CGM in the IP space with consistent responsiveness during 3 months in vivo. Future iterations of this system will utilize auto...

Observations of quantum dot (QD)–labeled cells in biomedical research are mainly qualitative in nature, which limits the ability of researchers to compare results experiment to experiment and laboratory to laboratory. Labeled cells are useful in a range of in vitro and in vivo assays where tracking behavior of administered cells is integral for answering research questions in areas such as tissue engineering and stem cell therapy. Before the full potential of QD‐based toolsets can be realized, uptake of QDs by cells must be quantified and standardized. This unit describes a novel, simple method to assess the number of QDs per cell using flow cytometry and commercially available standards. This quick and easy method can be used to calibrate flow cytometry instruments and settings, and quantify QD uptake by cells for in vitro and in vivo experimentation for comparable results across QD conjugate types, cell types, research groups, lots of commercial QDs, and homemade QDs. Curr. Protoc. Cytom. 49:6.26.1‐6.26.7. © 2009 by John Wiley & Sons, Inc.