Can you suggest a method to monitor cell viability in vitro using an immunofluorescence microscope from cell culture? I have PI and Texas Red, but I am not sure about the correct protocol. Thanks!
Hello Tugba Yalcinkaya
Since you have PI, I can help you with the protocol for cell viability in vitro using an immunofluorescence microscope.
1. Prepare PI stock solution.
Add 1mg of PI to 1ml of distilled water to prepare a stock solution of PI of 1mg/ml.
2. Dilute the PI stock solution in PBS to a final concentration of 10-50ug/ml. For instance, if you are to prepare 10ug/ml working solution, dilute 10ul of 1mg/ml stock solution in 990ul of PBS.
3. Harvest cells and resuspend them in an appropriate volume of buffer.
4. Add PI working solution to the cell suspension (say 5-10ul of PI staining solution per 100ul of cells).
5. Incubate the cells with PI for 10-15 minutes at 37 degree C.
6. Analyze the stained cells using fluorescence microscopy. PI when bound to DNA, has an excitation maximum around 535 nm and an emission maximum around 617 nm.
Just a suggestion. For more accurate viability assessment, you should consider using PI in combination with a live cell stain like calcein AM. Calcein AM is a cell-permeable, non-fluorescent derivative of calcein. Once inside live cells, calcein AM is hydrolyzed by intracellular esterases, releasing the fluorescent calcein. The resulting calcein molecule is a green-fluorescent dye, allowing for visualization of live cells.
When calcein AM is used in conjunction with PI, it is possible to determine viability based on the number of metabolically active (green fluorescent) and non-viable (red fluorescent) cells in the sample.
Before staining cells, you prepare a calcein AM stock solution by dissolving it in DMSO at a concentration of 1-5mM. Prepare a working solution (1-10uM) by diluting the stock solution. Use a small amount of the dye (approx. 1/10th of the culture medium volume) and incubate the cell suspension for 15 mins at 37 deg C. Calcein AM has excitation and emission wavelengths at around 494 nm and 517 nm, respectively.
Best,
Malcolm Nobre Thank you very much for your answer. It was very useful for me. Can I use PI to visualize cells in plate medium without damaging the cells?
Hello Tugba,
It should be taken into consideration that PI (Propidium Iodide) cannot penetrate the intact membrane of live cells, thus it can be used to distinguish between live and dead cells; whereas Texas Red is solely a fluorescent dye and does not have the capability to assess cell viability (i.e., it cannot detect cell viability status).
For convenient cell viability assessment, you may also consider PI-based assay kits, such as: Viability/Cytotoxicity Assay Kit for Live & Dead Cells (Calcein/PI) (MCE, HY-K1094)
https://www.medchemexpress.com/inhibitor-kit/viability-cytotoxicity-assay-kit-for-live-dead-cells-calcein-pi.html
If you need to consult for more experimental details, you are welcome to seek help from us at any time by following and sending private messages or leaving comments.
Hope this helpful!
The protocol for measuring cell viability using the Viability/Cytotoxicity Assay Kit for Live & Dead Cells (Calcein/PI) (MCE, HY-K1094) is attached below for your reference.
Pre-experiments to confirm the optimal concentration of staining solutions (Optional)
Since staining conditions vary depending on cell type and cell concentration, it is recommended to perform pre-experiments to determine the optimal concentrations of Calcein-AM and PI staining solutions.
1. Dead Cells preparation: Incubate cells in 70% ethanol for 30 min or in 0.1% saponin for 10 min.
Note: The method for preparing dead cells is not fixed. Alternative methods, such as treatment with 0.1%-0.5% digitonin for 10 min, can also be used.
2. Reagent Preparation: Remove the PI and Calcein-AM staining solutions to equilibrate at room temperature for 30 min.
3. PI Staining: Prepare several concentrations of PI working solutions (0.1-10 μM) using the assay buffer and stain the dead cells. Identify the concentration of PI that stains the nuclei without staining the cytoplasm.
4. Calcein-AM Staining: Prepare several concentrations of Calcein-AM working solutions (0.1-10 μM) using the assay buffer and stain dead cells. Identify the optimal Calcein-AM concentration that does not stain the cytoplasm. Subsequently, use this concentration to stain live cells and verify that live cells can be successfully stained.
Note: It is generally recommended to use the lowest possible dye concentration that provides sufficient signal intensity for the experiment.
Preparation of Staining Working Solution
The recommended staining concentrations for PI and Calcein-AM are 0.1–10 μM. The optimal concentration can be determined based on pre-experimental results.
The following example uses 2 μM Calcein-AM and 8 μM PI:
1. Reagent Preparation: Remove the PI and Calcein-AM staining solutions and equilibrate at room temperature for 30 min.
2. Preparation of Staining Working Solution: Add 5 μL of PI staining solution and 5 μL of Calcein-AM staining solution into 10 mL of assay buffer. Mix thoroughly to obtain the staining working solution, which can be used directly for staining.
Staining
Adherent Cells
1. Seed adherent cells into cell culture plates, microplates, or prepare cell coverslips.
Note: Suspended cells can also be prepared as cell coverslips.
2. After treating the cells according to the experimental design, wash the cells 2–3 times with PBS to completely remove residual active esterases in the culture medium.
3. Add an adequate amount of staining working solution, ensuring that the monolayer cells are fully covered.
4. Incubate at 37°C for 15 - 30 min.
Suspended Cells
1. After treating the cells according to the experimental design, centrifuge at 1,000 rpm for 3 min, discard the supernatant, and collect the cells.
Note: Recommended cell quantity is 1 × 104 - 1 × 105.
2. Wash the cell pellet 2 - 3 times with PBS to completely remove residual active esterases in the culture medium.
3. Resuspend the cell pellet in 100 μL of staining working solution, ensuring a cell density to 1 × 105 - 1 × 106 cells/mL。
4. Incubate at 37°C for 15 - 30 min.
Fluorescence Detection and Analysis
Fluorescence Microscopy Detection
1. Adherent Cells: For cells in culture plates, aspirate the staining working solution to stop the staining. Wash the cells 2-3 times with PBS, and add a sufficient amount of assay buffer to completely cover the monolayer cells for observation. For cell coverslips, add 10 μL of assay buffer to a clean microscope slide to fully cover the coverslip for observation.
Note: Cell coverslips can be sealed with nail polish to prevent evaporation.
2. Suspension Cells: Add 10 μL of the stained cell suspension to a clean microscope slide. Seal with nail polish to prevent evaporation.
3. Detection: Use a fluorescence microscope with an excitation wavelength of 490 ± 10 nm to simultaneously observe live cells (yellow-green fluorescence) and dead cells (red fluorescence). Additionally, use an excitation wavelength of 545 nm to observe dead cells alone.
Fluorescence Microplate Reader Detection
1. Control Setup: Prepare control and experimental groups following the staining working solution preparation and staining steps.
Experimental Groups: A, B
Control Groups: Dead Cell Controls (C, D), Live Cell Controls (E, F), and Cell-Free Controls (G, H).
Note: Dead cell control can refer to the pre-experimental dead cell preparation method.
2. Adherent Cells: Can be directly detected.
3. Suspension Cells: Seed 100 μL of the stained cell suspension per well in a microplate.
Note: The minimum detectable cell count per well is 200 - 500 cells, and the maximum is 1 × 106 cells.
4. Detection: Configure appropriate excitation and emission wavelengths to collect data.
For Calcein-AM: Excite at 490 ± 10 nm and collect emission signals at 530 ± 12.5 nm.
For PI: Excite at 530 ± 12.5 nm (typical Rhodamine optical filter) and collect emission signals at 645 ± 20 nm.
Note: It is recommended to use a microplate reader with optical filters to ensure signal interference is minimized.
5. Analysis and Calculation
Define the percentage of live and dead cells based on fluorescence readings.
Absolute live and dead cell counts: Generate standard curves of cell counts versus fluorescence readings (530 nm and 645 nm). Fluorescence intensity correlates linearly with cell count in the sample.
Note: Dead cells exhibit strong signals at 645 nm and weak signals at 530 nm.
Flow Cytometry Analysis
For both suspension cells and trypsin-dissociated adherent cells, follow the above staining working solution preparation and staining steps. The stained cell suspension can be directly analyzed using flow cytometry.
Lucian Miles Your comment was very explanatory. Thank you. I will contact you again. Have a nice work.
Tugba Yalcinkaya, no, PI cannot be used to visualize cells. PI does not permeate viable cell membranes but passes through disturbed cell membranes and stains the nuclei. So, PI is often used in combination with a fluorescein compound, such as Calcein-AM for simultaneous staining of viable and dead cells.