Limits...
Live single cell functional phenotyping in droplet nano-liter reactors.

Konry T, Golberg A, Yarmush M - Sci Rep (2013)

Bottom Line: While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations.This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells.Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences School of Pharmacy Bouvé College of Health Sciences, Northeastern University, 140 The Fenway, Room 156, 360 Huntington Avenue Boston, Massachusetts 02115.

ABSTRACT
While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surface and secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

Show MeSH
(a) DCs and bead-based sensors and reagents co-encapsulated in nano-liter reaction droplet containing anti-CD86 fluorescently tagged Abs for cell surface analysis. 1,2. Fluorescence images of droplets captured on a Zeiss 200 Axiovert microscope (a1 × 20 and a2 × 40) after 2 hours and 20 min of incubation. 3. The intensity of the fluorescence recorded for both CD86 protein and IL-6 detection measured using ImageJ software (the fluorescence intensity scale is present on the right). (b, c, d) Physical interaction and the polymerization of tubulin in live DCs that encountered a T cell and establishing signaling zone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3822379&req=5

f2: (a) DCs and bead-based sensors and reagents co-encapsulated in nano-liter reaction droplet containing anti-CD86 fluorescently tagged Abs for cell surface analysis. 1,2. Fluorescence images of droplets captured on a Zeiss 200 Axiovert microscope (a1 × 20 and a2 × 40) after 2 hours and 20 min of incubation. 3. The intensity of the fluorescence recorded for both CD86 protein and IL-6 detection measured using ImageJ software (the fluorescence intensity scale is present on the right). (b, c, d) Physical interaction and the polymerization of tubulin in live DCs that encountered a T cell and establishing signaling zone.

Mentions: To measure the time dependent CD86 and IL-6 expression of single DCs, the fluorescent signal generated on cell and bead surfaces was recorded each 20 min in the confined volume of droplets and analyzed by the ImageJ software (Fig. 2a). The positive signal was recorded and quantified for each cell in the droplets, and compared to the background empty droplet signal (Fig. 2b). The image sequence was analyzed and the increase in intensity from each cell was plotted over time showing expression kinetics (Fig. 3a, b). After 40 minutes of incubation in LPS nano-liter droplets, the encapsulated cells started to upregulate the surface maturation marker, CD86, and reached saturation at 2 hours and 20 min (Fig. 3a). Fig. 3c describes the fluorescent signal intensity from thirty cells after 2 hours and 20 min in separate droplets, confirming the previously-described inherent CD86 marker heterogeneity1112131415161718. Thus, this method allows us to distinguish the responses of individual cells to LPS stimulation in the confined volume of droplets. In addition to CD86, we were able to monitor LPS induced IL-6 secretion from the same encapsulated single DCs (Fig. 3b). The fluorescence signal for the detection of IL-6 on microsphere surfaces is shown in Figure 2b for single DCs. The signal was detected in droplets after 10 min in the array (Figure 3c). Droplets with LPS stimulation contained IL-6 levels that were significantly higher (three standard deviations) than droplets with no LPS, which showed negligibleIL-6 levels.


Live single cell functional phenotyping in droplet nano-liter reactors.

Konry T, Golberg A, Yarmush M - Sci Rep (2013)

(a) DCs and bead-based sensors and reagents co-encapsulated in nano-liter reaction droplet containing anti-CD86 fluorescently tagged Abs for cell surface analysis. 1,2. Fluorescence images of droplets captured on a Zeiss 200 Axiovert microscope (a1 × 20 and a2 × 40) after 2 hours and 20 min of incubation. 3. The intensity of the fluorescence recorded for both CD86 protein and IL-6 detection measured using ImageJ software (the fluorescence intensity scale is present on the right). (b, c, d) Physical interaction and the polymerization of tubulin in live DCs that encountered a T cell and establishing signaling zone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3822379&req=5

f2: (a) DCs and bead-based sensors and reagents co-encapsulated in nano-liter reaction droplet containing anti-CD86 fluorescently tagged Abs for cell surface analysis. 1,2. Fluorescence images of droplets captured on a Zeiss 200 Axiovert microscope (a1 × 20 and a2 × 40) after 2 hours and 20 min of incubation. 3. The intensity of the fluorescence recorded for both CD86 protein and IL-6 detection measured using ImageJ software (the fluorescence intensity scale is present on the right). (b, c, d) Physical interaction and the polymerization of tubulin in live DCs that encountered a T cell and establishing signaling zone.
Mentions: To measure the time dependent CD86 and IL-6 expression of single DCs, the fluorescent signal generated on cell and bead surfaces was recorded each 20 min in the confined volume of droplets and analyzed by the ImageJ software (Fig. 2a). The positive signal was recorded and quantified for each cell in the droplets, and compared to the background empty droplet signal (Fig. 2b). The image sequence was analyzed and the increase in intensity from each cell was plotted over time showing expression kinetics (Fig. 3a, b). After 40 minutes of incubation in LPS nano-liter droplets, the encapsulated cells started to upregulate the surface maturation marker, CD86, and reached saturation at 2 hours and 20 min (Fig. 3a). Fig. 3c describes the fluorescent signal intensity from thirty cells after 2 hours and 20 min in separate droplets, confirming the previously-described inherent CD86 marker heterogeneity1112131415161718. Thus, this method allows us to distinguish the responses of individual cells to LPS stimulation in the confined volume of droplets. In addition to CD86, we were able to monitor LPS induced IL-6 secretion from the same encapsulated single DCs (Fig. 3b). The fluorescence signal for the detection of IL-6 on microsphere surfaces is shown in Figure 2b for single DCs. The signal was detected in droplets after 10 min in the array (Figure 3c). Droplets with LPS stimulation contained IL-6 levels that were significantly higher (three standard deviations) than droplets with no LPS, which showed negligibleIL-6 levels.

Bottom Line: While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations.This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells.Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences School of Pharmacy Bouvé College of Health Sciences, Northeastern University, 140 The Fenway, Room 156, 360 Huntington Avenue Boston, Massachusetts 02115.

ABSTRACT
While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surface and secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

Show MeSH