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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 lab-to-lab to improve the state-of-the-art. 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 in the clinic, 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 by all researchers to calibrate their 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
Without reliable, standardized quantitative information on quantum dot (QD) uptake by cells, researchers will not be able to conduct the dosage studies required to optimize QDs for biomedical and clinical applications, and most importantly, minimize QD toxicity if any — an area still under investigation. (Dubertret, Skourides et al. 2002; Medintz, Uyeda et al. 2005; Michalet, Pinaud et al. 2005; Hardman 2006; Jamieson, Bakhshi et al. 2007) A standardized, quantitative characterization of QD uptake will also facilitate optimization of QD conjugate chemistry to decrease batch-to-batch or lot-to-lot variance and resultant experiment-to-experiment variance currently plaguing the field. (Wu, Campos et al. 2007) This unit describes a novel, quick and easy method which can become the standard for determining a quantitative value for QD uptake by cells in units of number of QDs per cell.
Flow cytometry, unlike other methods to measure QD uptake such as elemental methods (inductively coupled plasma mass spectroscopy (ICP-MS), atomic emission mass spectroscopy (AES-MS)), or radiolabelling ((micro positron emission tomography (microPET)), is routinely used in cell biology and clinical labs and is nondestructive to samples. Commercially available fluorescent calibration beads are a standard for quantitative flow cytometry allowing direct and objective comparison of data between laboratories. (Wu, Campos et al. 2007) First, this unit will describe a protocol for developing a calibration curve for the flow cytometry instrument by titrating commercially available calibration beads with commercially available QDs. Second, this unit will describe the method by which one can use the calibration curve to determine the approximate number of QDs per cell for a given cell-labeling study.
In this protocol, the method for calibrating the flow cytometer to obtain quantitative information on QD uptake by cells is described.
This protocol goes through the steps required to quantify the approximate number of QDs taken up by cells in a given cell study using the calibration curve derived above. Cells can be labeled by QDs conjugated with polyArginine (polyArg), cholera toxin B (CTB), transferrin (TF), or other reported methods. (Lagerholm, Wang et al. 2004; Mattheakis, Dias et al. 2004; Chakraborty, Fitzpatrick et al. 2007)
Dissolve 1 TPS packet in 1 L deionized water. Dissolve appropriate mass of BSA in appropriate volume of TPS buffer by mixing with stir bar for 10 minutes. Filter solution through 0.22 μm filter unit. Store buffer for no more than one day at 4°C.
Over the past 10 years since quantum dots (QDs) were rendered biochemically stable for biological applications, (Bruchez, Moronne et al. 1998; Chan and Nie 1998) many groups have been working to optimize — or have already applied — QD conjugates to answer biological, biomedical, and clinical research questions. (Medintz, Uyeda et al. 2005; Michalet, Pinaud et al. 2005; Jamieson, Bakhshi et al. 2007) QDs are semiconductor nanoparticles that have size and composition adjustable fluorescence emission wavelengths, narrow emission bands, and very high levels of brightness and photostability. (Cai, Chen et al. 2007) In biomedical research, QDs have potential as a tool for cell labeling, fluorescence in situ hybridization, cell tracking in vitro and in vivo, fluorescence resonance energy transfer, cancer diagnosis, and tumor targeting in vivo and more. (Gao, Cui et al. 2004; Lagerholm, Wang et al. 2004; Medintz, Uyeda et al. 2005; Michalet, Pinaud et al. 2005; Cai, Shin et al. 2006; Fischer, Liu et al. 2006; Cai, Chen et al. 2007; Chakraborty, Fitzpatrick et al. 2007; Jamieson, Bakhshi et al. 2007; Yezhelyev, Al-Hajj et al. 2007) However, one of the most significant obstacles to advancement of the technology in the particular case of cell tagging and tracking is that there is no clear standard by which researchers can compare results of QD uptake by cells in vitro and between labs. (Hardman 2006)
Literature often reports cellular uptake of QDs by microscopy in qualitative terms such as dimmed, low, moderate, or high fluorescence, brightness, percentage, or presence of QDs in few, some, or many cells. Many studies simply state the initial loading concentration of QDs per cell, which is not reflective of the number of QDs taken up by cells. Experimental conditions which affect cellular uptake vary from lab-to-lab. Only one study by Dubertret et al. has reported a measured yet approximate, quantitative value of QD uptake in terms of number of QDs per cell. However, their work involved the use of injection as a delivery method rather than internalization by QD conjugates. (Dubertret, Skourides et al. 2002; Hardman 2006) Another group reports an observation of 1 million QDs per cell after 1 hr incubation with QD conjugates and 3 million QDs per cell with no supporting data, methodology or reference as to how these values were obtained. (Gao, Cui et al. 2004)
The method presented here is simple, quantitative and reliable. We believe that this method will allow direct comparison of methods and materials developed in distinct laboratories, provided an analogous material is available, or can be prepared as a mouse antibody conjugate.
Prior to flow cytometer calibration and cell studies, measure the absorbance and fluorescence of QD stock materials. Absorbance readings can be used to calculate a more precise concentration of QD stock solutions from the commercial source which will give a more accurate quantification of the number of QDs per cell based on these stock concentrations. In our experience, fluorescence values will show the extent of fluorescence emission variance between QD conjugates and batches. Since the calibration curve is created with one conjugate and is used to characterize another conjugate, it is important to factor in variability in fluorescence emission or brightness (which is related to quantum yield and extinction coefficient) between conjugates and batches of QDs.
QSC bead kits contain five populations of polystyrene microbeads that have a predetermined, progressively increasing number of ligands on each bead (bead diameter of 8 μm). Lot-to-lot, kits vary in the binding capacity of given populations, therefore signal at saturation or Bmax will also vary lot-to-lot.
Bead populations “ABC0” and “ABC1” should not be used for calibration as they both exhibited nonspecific binding behavior in protocol development studies regardless of efforts to optimize buffer composition. Instead, a nonspecific binding assay was developed for each bead population where beads are blocked with free IgG prior to titration with IgG QDs.
For successful calibration, the flow cytometer voltage settings must be adjusted such that cells loaded with reasonable concentrations of QDs are visible at the same settings used for the titration of QSC beads (settings used for the calibration).
Another variance by batch of beads is the number of beads in suspension provided by the commercial source. This concentration of particles will affect how much volume of beads you will need to titrate in order to read at least 5,000 events on your flow cytometer.
If there is a lot of visible, non-bead events seen in the scatter plot during flow cytometric analysis of QD IgG bound beads, this means that there is a significant amount of junk in stock QD materials. Spin down the QD stock from the commercial source at 5,000 rcf for 5 minutes and avoid bottom of vial, or set a threshold FSC on events to decrease the amount of debris counted as events in flow cytometry.
An example calibration curve generated for 705 nm emitting QDs is shown in Figure 1 and includes two batches of QSC bead sets. The R2 value for the linear regression of the data was good at 0.9775. For this example calibration curve, any signal from cells run on the flow cytometer, signal, in terms of reference standard units (signal of the sample divided by the signal of the reference standard) can be related to number of QDs per cell, No.QDs/Cell, according to the following equation,
The calibration curve generated in this protocol remains linear and holds true through the limit of detectable signal on the cytometer being used. For example, the limit of the cytometer used in development of this protocol is 100,000 arbitrary units, corresponding to approximately 1,000 reference standard units or 200 million QDs. In studies conducted during the development of this protocol, the largest average number of QDs detected per cell was 2.4 million, with some events as high as 100 million QDs per cell, in a dendritic cell line for a loading concentration of 8 nM QDs. In literature, cells are typically stained with QDs at a loading concentration of 1,000 pM to 10 nM. Therefore, it is reasonable that a sensitivity limit of 200 million QDs per cell using this calibration curve and cytometer settings falls far beyond the physical capacity of cells for QDs. In fact, this value is near the capacity of mammalian cells for QDs assuming the cell’s entire volume is completely filled with QDs.
The binding titration assay for calibration curve development takes about 2.5 hours. Flow cytometry analysis of these titrated samples takes about 4 hours. Data analysis of flow data in software such as FlowJo takes about 1 hour and fitting resultant data to curves in Graphpad Prism software takes about 1 hour for the experienced user. Deriving the calibration curve in spreadsheet software such as Microsoft Excel takes a few minutes.
Preparing cells for flow cytometry analysis takes about 1.5 hours and actual analysis on the flow cytometer takes 1 hour. Analysis of cell flow data in flow analysis software and quantification of number of QDs per cell in spreadsheet software using the developed calibration curve takes about 1 to a few hours depending on the number of samples of the cell study.