Nexcelom Bioscience
Cell Viability Trypan Blue AOPI Cellometer Applications

Using Trypan Blue and Acridine Orange/Propidium Iodide to Measure Cell Viability

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Introduction

Since the quality of the cell sample is vital for potential downstream experiments, viability measurements are routinely performed in many laboratories. Choosing the correct method for conducting cell viability measurement is essential for obtaining consistent accurate results. Here we review two common methods for measuring cell viability: Trypan Blue, and Acridine Orange/Propiduim Iodide.

Using Trypan Blue to Measure Cell Viability

One of the earliest and most common methods for measuring cell viability is the trypan blue (TB) exclusion assay. Trypan blue is a ~960 Daltons molecule that is cell membrane impermeable and therefore only enters cells with compromised membranes. Upon entry into the cell, trypan blue binds to intracellular proteins thereby rendering the cells a bluish color. The trypan blue exclusion assay allows for a direct identification and enumeration of live (unstained) and dead (blue) cells in a given population. Although trypan blue has been used to determine cell viability for many years, it is not without its drawbacks. It is considered to be carcinogenic and must be handled with care and disposed of properly. Over time trypan blue naturally forms dye aggregates and crystals, it is therefore recommended that TB is filtered using a 0.2 micron filter prior to use. Finally, multiple publications have observed that trypan blue viability measurements in samples that are lower than 70 % viable show higher measured cell viability when compared to fluorescent based detection methods 1,2. Data suggests that conducting viability measurements with trypan blue on samples with low viability may not be optimal. We recommend that trypan blue assay is ideally used for cultured cell lines and purified/isolated cell samples with viabilities greater than 70 %.

Jurkat Cells Stained with Trypan Blue

Trypan blue live and dead cells

Using Acridine Orange/Propiduim Iodide (AO/PI) to Measure Cell Viability

Acridine orange (AO) and propidium iodide (PI) are nucleic acid binding dyes that can be used to measure the cell viability. Both AO and PI are considered skin irritants and may be harmful if ingested or inhaled; they are currently not classified as carcinogenic. Since AO is cell permeable, all stained nucleated cells generate a green fluorescence. PI (~668 Daltons) only enters cells with compromised membranes and therefore dying, dead, and necrotic nucleated cells stained with PI generate a red fluorescence. When cells are stained with both AO and PI, live nucleated cells only fluoresce green and dead nucleated cells only fluoresce red. This is due to Förster resonance energy transfer (FRET); where the PI signal absorbs the AO signal producing no spill-over or double positive results. Additionally, other membrane-exclusion viability dyes such as: ethidium bromide (EB), 7AAD, SYTOX green/red, DRAQ5 and others may also be used instead of PI. Cell viability is calculated by examining the ratio of the number of live to the number of dead fluorescing cells. This assay can not only be used to measure the viability of nucleated cells in cell culture and purified samples but also in complicated samples such as PBMC, whole blood, bone marrow, bronchoalveolar lavage, tumor digests, primary samples and many more.

Bright field image of a peripheral blood sample

Bright field image of a peripheral blood sample

Live nucleated cells stained with Acridine Orange

Live nucleated cells stained with Acridine Orange

Dead nucleated cells stained with Propidium Iodide

Dead nucleated cells stained with Propidium Iodide

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Trypan Blue Protocol: Sample Preparation and Analysis

Simple, User-friendly Cellometer Procedure

Using Cellometer Mini, Auto 1000, Auto T4, Auto 2000, K2 and Vision

Simple, User-friendly Procedure

1. Pipette 20µl

2. Insert Slide

3. Select Assay & Click Count

4. Results in 30 seconds!

Trypan Blue Protocol

  1. Dilute the stock (0.4 %) with PBS to 0.2 %.
  2. Filter the trypan blue with 0.2 micron filter
  3. Mix the cell suspension at 1:1 with 0.2 % trypan blue
  4. Load the counting chamber slide into the Cellometer and analyze

Viability Measurement of Purified Primary Cell Lines and Cell Culture Using Trypan Blue

Viability measurement of Jurkat cells by trypan blue 

Jurkat cells imaged by Cellometer Vision

 
Viability measurement of CHO cell by trypan blue 

CHO cells imaged by Cellometer Vision

 
Viability measurement of HeLa cells by trypan blue 

HeLa cells imaged by Cellometer Auto T4

 
Viability measurement of splenocyte cells by trypan blue 

Splenocytes imaged by Cellometer Auto 2000

 
Viability measurement of Jurkat cell by trypan blue 

Jurkat cells imaged by Cellometer Auto 1000

 

The micrographs displayed above represent cultured cells and purified primary cells that have been stained with trypan blue and analyzed on the Cellometer instruments. The dark cells delineated with red circles represent dead trypan blue positive cells. Cells with bright centers are considered live.

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AO/PI Protocol: Sample Preparation and Analysis

Simple, User-friendly Cellometer Procedure

Using Cellometer Vision, Auto 2000 and K2

With the Cellometer Vision, Auto 2000, and K2 just 20µl of sample is added to the Cellometer Counting Chamber. Imaging and analysis of the samples is completed in less than 30 seconds. Bright field and fluorescent cell images can be viewed to check cell morphology and verify cell counting. Total cell count, concentration, and mean diameter are automatically displayed.

AO/PI Protocol

  1. Obtain Nexcelom AO/PI solution: Cat # CS2-0106-5ML.
  2. Stain cell sample at 1:1 with AO/PI solution
  3. Load counting chamber slide and analyze
Pipette 20 µl of cell sample into a disposable counting slide

1. Pipette 20 µl of cell sample into a disposable counting slide.

Insert slide in to cell counter

2. Insert slide into the instrument

select AO/PI assay

3. Select assay from a drop down menu

cell count, concentration, and diameter

4. Click count, acquire image and view cell count, concentration, diameter

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Viability Measurement for Messy Samples Using AO/PI


Tumor digest sample

Tumor Digest Samples

Tumor digest samples are collected from the patient at the primary source of the tumor. Solid tumors are first digested and processed before they are utilized in experiments. These are inherently messy samples. They may contain tissue fragments and cellular debris making it difficult to identify and count cells of interest. With AO/PI only the nucleated cells are stained and analyzed

Leuko Pak

Leuko Pak

Cord Blood

Cord Blood

Both Leuko Pak and Cord blood samples contain a large amount of red blood cells, platelets, cell debris making it impossible to count in brightfield. Using AO/PI the concentration and viability of mononuclear cells can be measured.

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Conclusions & Cellometer Selection Guide

The Cellometer line of instruments can quickly and accurately obtain the viability of the cell sample by trypan blue or fluorescent methods. Additionally, the system automatically reports an accurate cell count, concentration, and cell size in a single 20 µl assay. The fluorescent based Cellometer instruments (Auto 2000, Vision, and K2) also allow researcher to count the total number of nucleated cells in a fresh clinical sample without the need to lyse red blood cells. The table below outlines the recommended Cellometer instrument and assay use for each selected application.

 
Cellometer Mini
Cellometer Auto T4
Cellometer Auto 1000
Cellometer Auto 2000
Cellometer Vision / Vision CBA
Purified cells– no debris
TB
TB
TB
TB or AOPI
TB or AOPI
Isolated MNCs – without lysing RBCs
NR
NR
NR
AOPI
AOPI
Fresh BM, CB, WB, MNCs w/ lysing of RBCs
TB
TB
TB
TB or AOPI
TB or AOPI
Fresh BM, CB, WB without lysing
NR
NR
NR
AOPI
AOPI
Frozen BM, CB, WB
TB
TB
TB
TB or AOPI
TB or AOPI
Digested tumors and BAL with tissue/cellular debris
NR
NR
NR
AOPI
AOPI

AOPI = Acridine Orange/Prodidium Iodide, TB = Trypan Blue, NR = not recommended, BM = Bone Marrow, CB = Cord Blood, WB = Whole Blood, MNCs = Mononuclear cells

Please contact an application specialist to determine which Cellometer is right for you!

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Publications Using Cellometer Instruments for Trypan Blue Viability

  • Zierold C, Carlson MA, Taylor DA, et al. (2011) Developing mechanistic insights into cardiovascular cell therapy: Cardiovascular Cell Therapy Research Network Biorepository Core Laboratory rationale.  American Heart Journal 162(6): 973-80
  • Bruchova H, Yoon D, Prchal JT, et al. (2007) Regulated expression of microRNAs in normal and polycythemia vera erythropoiesis. Experimental Hematology 35(11): 1657-67
  • Duncan JA, Gao X, Ting JP, et al. (2009) Neisseria gonorrhoeae activates the proteinase cathepsin b to mediate the signaling activities of the nlrp3 and asc containing inflammasomes. Journal of Immunology 182(10): 6460-9
  • Xu W, Soga S, Neckers L, et al. (2007) Sensitivity of epidermal growth factor receptor and ErbB2 exon 20 insertion mutants to Hsp90 inhibition. British Journal of Cancer 97(6): 741-4
  • Dhar K, Dhar G, Banerjee S, et al. (2010) Tumor cell-derived PDGF-B potentiates mouse mesenchymal stem cells-pericytes transition and recruitment through an interaction with NRP-1. Molecular Cancer 9(8) :209
  • Staszkiewicz J, Frazier TP, Gawronska-Kozak B, et al. (2010) Cell Growth Characteristics, Differentiation Frequency, and Immunophenotype of Adult Ear Mesenchymal Stem Cells. Stem Cells Development 19(1): 83-92
  • Weil BR, Markel TA, Meldrum DR, et al. (2009) Mesenchymal stem cells enhance the viability and proliferation of human fetal intestinal epithelial cells following hypoxic injury via paracrine mechanisms. Surgery 146(2): 190-7
  • O'Connor CM, Sheppard S, Cooper LJ, et al. (2012) Adoptive T-cell therapy improves treatment of canine non-Hodgkin lymphoma post chemotherapy. Scientific Reports 2: 249
  • Karabulut B, Karaca B, Uslu R, et al. (2011) Regulation of apoptosis-related molecules by synergistic combination of all-trans retinoic acid and zoledronic acid in hormone-refractory prostate cancer cell lines. Molecular Biology Reports 38(1): 249-59
  • Farina A, D'Aniello C, Chambery A, et al. (2011) Temporal proteomic profiling of embryonic stem cell secretome during cardiac and neural differentiation. Proteomics 11(20): 3972-82
  • Singh H, Figliola MJ, Cooper LJ, et al. (2011) Reprogramming CD19-specific T cells with IL-21 signaling can improve adoptive immunotherapy of B-lineage malignancies. Cancer Research 71(10): 3516-27
  • Rose AA, Grosset AA,Siegel PM, et al. (2010) Glycoprotein Nonmetastatic B Is an Independent Prognostic Indicator of Recurrence and a Novel Therapeutic Target in Breast Cancer. Clinical Cancer Reseach 16(7): 2147-56
  • Kucukzeybek Y, Gorumlu G, Uslu R, et al. (2010) Apoptosis-mediated cytotoxic effects of ibandronic acid on hormone and drug-refractory prostate cancer cells and human breast cancer cells. Journal of International Medical Research  38(5): 1663-72
  • Ray S, Langan RC, Avital I, et al. (2012) Establishment of Human Ultra-Low Passage Colorectal Cancer Cell Lines Using Spheroids from Fresh Surgical Specimens Suitable for In Vitro and In Vivo Studies. Journal of Cancer 3: 196-206
  • Agarwal HK, Chhikara BS, Parang K, et al. (2013) Emtricitabine Prodrugs with Improved Anti-HIV Activity and Cellular Uptake. Molecular Pharmacology 10(2): 467-76
  • Ross AP, Webster TJ. (2013) Anodizing color coded anodized Ti6Al4V medical devices for increasing bone cell functions. International Journal of Nanomedicine 8: 109-17
  • Diep CH, Zucker KM, Han H, et al. (2012) Down-regulation of Yes Associated Protein 1 expression reduces cell proliferation and clonogenicity of pancreatic cancer cells. PLoS One 7(3): e32783
  • Guercio A, Di Bella S, Piccione G, et al. (2013) Canine Mesenchymal Stem Cells (MSCs): Characterization in Relation to Donor Age and Adipose Tissue-Harvesting Site. Cell Biology International 37(8): 789-98

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Publications Using Cellometer Instruments for AO/PI Viability

  • Chan LL, Zhong XM, Pirani A, Lin B. (2012) A novel method for kinetic measurements of rare cell proliferation using Cellometer image-based cytometry. Journal of Immunological Methods 377(1-2): 8-14
  • Chan LL, Wilkinson AR, Paradis BD, Lai N. (201) 2Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods. Journal of Fluorescence 22(5): 1301-1311
  • Holmuhamedov EL, Czerny C, Beeson CC, Lemasters JJ. (2012) Ethanol Suppresses Ureagenesis in Rat Hepatocytes Role of Acetaldehyde. Journal of Biological Chemistry 287(10): 7692-7700
  • Ranguelova K, Rice AB, Khajo A, et al. (2012) Formation of reactive sulfite-derived free radicals by the activation of human neutrophils: An ESR study. Free Radical Biology & Medicine 52:1264-1271
  • Lugli E, Gattinoni L, Roberto A, et al. (2012) Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cells. Nature Protocols 8(1): 33-42
  • Chan LLY, Laverty DJ, Smith T, et al. (2013) Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error. Journal of Immunological Methods 388(1-2): 25-32

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References Comparing Trypan Blue to AO/PI for Viability Detection

  1. Altman SA, Randers L, Rao G. (1993) Comparison of Trypan Blue Dye Exclusion and Fluorometric Assays forMammalian Cell Viability Determinations. Biotechnology Progress 9(6): 671-674
  2. Mascotti K, McCullough J, Burger SR. (2000) HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion 40(6): 693-696

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