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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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

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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%.

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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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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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For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.© 2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Printed in the USA. CO23168 0611

Ordering informationProduct Cat. No.

CellLight® Histone 2B-GFP (BacMam 2.0) C10594

CellLight® Mitochondria-GFP (BacMam 2.0) C10600

CellLight® Mitochondria-RFP (BacMam 2.0) C10601

CellLight® Golgi-GFP (BacMam 2.0) C10592

CellLight® Peroxisome-GFP (BacMam 2.0) C10604

BacMam GFP Transduction Control (BacMam 2.0) B10383