GFP DETECTION Attune ® Acoustic Focusing Cytometer Detecting Green Fluorescent Protein using the Attune ® Acoustic Focusing Cytometer Flow cytometry has been used widely in cellular biology for analysis of fluorescent proteins. BacMam CellLight ® reagents are fluorescent protein–signal peptide fusions that provide accurate and specific targeting to cellular structures for live-cell applications, here used with human osteosarcoma (U2OS) cells. In this study, we demonstrate the ability to detect Green Fluorescent Protein (GFP) with the Attune ® Acoustic Focusing Cytometer, showing correlative imaging results, and also demonstrate the ability of the Attune ® Cytometric Software to easily create overlay plots. Basic protocol U2OS cells (1 x 10 5 ) were plated in separate 10 cm tissue culture dishes in complete medium. Including an unlabeled control, various GFP-expressing BacMam CellLight ® reagents were added at 2% v/v and incubated for 24 hr (Figures 1 and 2). In parallel, cells were plated into 6 separate dishes and labeled with histone 2B (H2B)–GFP at concentrations of 10%, 5%, 2%, 1%, 0.5%, and 0.2% v/v, and incubated for 24 hr (Figures 3 and 4). Figure 5 was generated from cells that were transduced with H2B-RFP and mitochondria-GFP simultaneously (2% v/v each). After 24 hr, all cells were imaged, harvested, and resuspended in PBS at a concentration of 1 x 10 6 cells/mL for analysis on the Attune ® Acoustic Focusing Cytometer. Samples were acquired and analyzed on the Attune ® cytometer using a 488 nm laser with 530/30 bandpass filters for GFP and 575/24 bandpass filters for RFP. The main population of cells was gated to exclude debris, and 50,000 gated events were collected at a rate of 200 µL/min in standard sensitivity mode. For single GFP results, histograms were generated and sample analysis with overlay plots was performed using the Attune ® Cytometric Software. A dual- parameter plot was generated for the dual-expressing GFP and RFP cells. Sensitive detection of GFP expression The Attune ® Acoustic Focusing Cytometer delivers rapid and sensitive analysis of GFP-expressing and GFP/RFP co-expressing cell populations, providing a quick and reliable method to quantitatively evaluate cell transfection with fluorescent proteins. Overlay plots can be made directly in the Attune ® Cytometric Software by using the simple drag-and-drop feature, where the legend displayed above the overlay plot has the names of each sample in the corresponding color.
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GFP DETECTIONAttune® Acoustic Focusing Cytometer
Detecting Green Fluorescent Protein using the Attune® Acoustic Focusing Cytometer
Flow cytometry has been used widely in cellular biology for analysis of fluorescent proteins. BacMam CellLight® reagents are fluorescent protein–signal peptide fusions that provide accurate and specific targeting to cellular structures for live-cell applications, here used with human osteosarcoma (U2OS) cells. In this study, we demonstrate the ability to detect Green Fluorescent Protein (GFP) with the Attune® Acoustic Focusing Cytometer, showing correlative imaging results, and also demonstrate the ability of the Attune® Cytometric Software to easily create overlay plots.
Basic protocolU2OS cells (1 x 105) were plated in separate 10 cm tissue culture dishes in complete medium. Including an unlabeled control, various GFP-expressing BacMam CellLight® reagents were added at 2% v/v and incubated for 24 hr (Figures 1 and 2). In parallel, cells were plated into 6 separate dishes and labeled with histone 2B (H2B)–GFP at concentrations of 10%, 5%, 2%, 1%, 0.5%, and 0.2% v/v, and incubated for 24 hr (Figures 3 and 4). Figure 5 was generated from cells that were transduced with H2B-RFP and mitochondria-GFP simultaneously (2% v/v each). After 24 hr, all cells were imaged, harvested, and resuspended in PBS at a concentration of 1 x 106 cells/mL for analysis on the Attune® Acoustic Focusing Cytometer.
Samples were acquired and analyzed on the Attune® cytometer using a 488 nm laser with 530/30 bandpass filters for GFP and 575/24 bandpass filters for RFP. The main population of cells was gated to exclude debris, and 50,000 gated events were collected at a rate of 200 µL/min in standard sensitivity mode. For single GFP results, histograms were generated and sample analysis with overlay plots was performed using the Attune® Cytometric Software. A dual-parameter plot was generated for the dual-expressing GFP and RFP cells.
Sensitive detection of GFP expressionThe Attune® Acoustic Focusing Cytometer delivers rapid and sensitive analysis of GFP-expressing and GFP/RFP co-expressing cell populations, providing a quick and reliable method to quantitatively evaluate cell transfection with fluorescent proteins. Overlay plots can be made directly in the Attune® Cytometric Software by using the simple drag-and-drop feature, where the legend displayed above the overlay plot has the names of each sample in the corresponding color.
Figure 1. U2OS cells labeled with various GFP-expressing BacMam CellLight® reagents and measured with the Attune® cytometer. Overlay plots, made with the Attune® Cytometric Software, display unlabeled cells and the transduced GFP-expressing cells. (A) Cells labeled with histone-2B GFP are divided into a large population of cells expressing GFP and a smaller segment that exhibits fluorescence slightly brighter than the unlabeled control. (B) Cells labeled with mitochondria-GFP separate into a large population of cells brightly expressing GFP and a very small population dimly expressing GFP (positioned to the right of the main peak next to the unlabeled sample). (C) Cells labeled with cytoskeleton-GFP show almost equal numbers of bright and dim GFP-expressing cells. (D) Cells labeled with golgi-GFP reveal a majority of the population to be expressing GFP and a very small population of dimly expressing cells. (E) Cells labeled with peroxisome-GFP exhibit a large, brightly fluorescent population and a minor population of dim GFP-expressing cells.
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H2B-GFP
Golgi-GFP
Mito-GFP
Peroxi-GFP
Cyto-GFP
Figure 2. Visual confirmation of GFP expression in cells. Prior to analysis on the Attune® Acoustic Focusing Cytometer, samples of each of the transduced cell populations were viewed using fluorescence microscopy to confirm GFP expression. (A) Histone-2B GFP; (B) Mito-GFP; (C) Cyto-GFP; (D) Golgi-GFP; and (E) Peroxi-GFP. Images were generated on a Zeiss microscope using 10x magnification and 100 ms.
A. Histone-2B B. Mitochondria C. Cytoskeleton D. Golgi E. Peroxisome
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GFP fluorescence
GFP fluorescence
GFP fluorescence
GFP fluorescence
GFP fluorescence
Figure 4. Visual confirmation of GFP expression from dilution series transduction. Prior to analysis on the Attune® Acoustic Focusing Cytometer, samples of each of the transduced cell populations were viewed using fluorescence microscopy to confirm GFP expression. Images were generated using a Zeiss microscope with an Alexa Fluor® 488 dye filter (10x magnification and 100 ms). (A) 10%, (B) 5%, (C) 2%, (D) 1%, (E) 0.5%, and (F) 0.2%.
A. 10%
D. 1%
B. 5%
E. 0.5%
C. 2%
F. 0.2%
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GFP fluorescence
Figure 5. U2OS cells labeled with BacMam CellLight® GFP and RFP reagents and measured with the Attune® Acoustic Focusing Cytometer. U2OS cells were simultaneously labeled with H2B-GFP and Mito-RFP at 2% v/v each. (A) A dual-parameter Attune® cytometer plot shows that the majority of the population is co-labeled with both RFP and GFP. (B) The same sample was imaged prior to flow analysis and confirms co-expression of both fluorescent proteins. The image was generated using a Zeiss microscope with 50 ms GFP and 250 ms RFP, 10x magnification.
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Mitochondria-RFP fluorescence
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-GFP
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Figure 3. U2OS cells labeled with H2B-GFP BacMam CellLight® reagent at various concentrations and measured with the Attune® Acoustic Focusing Cytometer. The overlay plot, made with the Attune® Cytometric Software, displays unlabeled cells and the transduced GFP-expressing cells. U2OS cells transduced with a dilution series of H2B-GFP BacMam CellLight® reagents showed corresponding levels of GFP expression, from brightest to dimmest as follows: 10% v/v (navy), 5% v/v (gray), 2% v/v (aqua), 1% v/v (pink), 0.5% v/v (green), 0.2% v/v (red), and untransduced (black). An inverse relationship exists where the unstained population for each transduction increases as the amount of CellLight® reagent introduced decreases. Fluorescence quantitation data were obtained from analysis of the various cell populations using the Attune® Acoustic Focusing Cytometer.
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