CMtO Protocols
Steps to Success in optimizing your samples for Cytometry and Microscopy to Omics
- Sample preparation – how to prepare a single cell suspension depending on your starting sample; viability tips for the healthiest sample.
- Staining considerations – basic steps and tips provided for cell surface, intracellular or nuclear staining; fixation and permeabilization tips
- Dead cell discrimination – explanation of importance, examples of fixable and unfixable dyes
- Doublet exclusion – avoid false positives
DNA Content
In order to measure the genome size of an unknown species the control should be made out of non-diving cells with nucleus. In order to measure DNA content of mammalian cells, chicken (Gallus gallus domesticus) erythrocytes (1.25 pg) or nuclei from human inactivated leukocytes (7.0 pg) are commonly used. Most popular fluorochromes are propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI). DAPI was found to be good for staining because it binds specifically to double-stranded DNA (dsDNA) independent of the chromatin structure, resulting in peaks with a low coefficient of variation (CV) but it binds preferentially to AT-rich portions of the dsDNA [Zhu et al. 2012]. It is generally agreed that fluorochromes showing base preference (DAPI) should NOT be used for estimation of nuclear DNA content in cases where base content of a control and a sample differ [Doležel et al. 1992].
Genome size quantified by the PI-based flow cytometry measurements are considered to be as good as that measured by the Feulgen microspectrophotometry (method for determining the amounts of DNA in individual cell nuclei).
After performing flow cytometry measurements nuclear DNA content can be calculated using this formula if using Glycine max as your control:
After staining the cells and following flow cytometry analysis, simple calculation based on samples’ histograms will determine your samples’ DNA content. If using chicken RBCs:
x – fluorescence channel number of your unknown sample
y – fluorescence channel number of chicken RBC
After calculating mean number DNA content one copy of genetic information (1C) can be determined by considering that 1 pg DNA equals 0.978*109 bp [Doležel et al., 2002].
Control = Glycine max (2C = 2.5 pg). Unknown samples which nuclei were stained with PI [Musa (a-e) and Ensete (f) ]. Their relative DNA content was measured based on G0/G1 peak positions: (a) “Pisang Mas” (2C = 1.243 pg); (b) “Cameroun” (2C = 1.130 pg); (c) M. textilis (2C = 1.435 pg); (d) M. beccarii (2C = 1.561 pg) and (e) M. ornata (2C = 1.299 pg); (f) E. gilletii (2C = 1.210 pg). Picture obtained from Bartoš et al. 2005
Protocols
Protocols for staining whole cells with PI and PI/ Bromodeoxyuridine
Analysis of Nuclear DNA Content and Ploidy in Higher Plants
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.
Cell Cycle Analysis
The Cytometry and Microscopy to Omics now offers an online MultiCycle AV add on to our FCS Express analysis program. Please contact the facility if you need help using MultiCycle.
Below are some examples of cell cycle data acquired using a BD LSR II flow cytometer and analysed with FCS Express software.
FCS Express Analysis
| Diploid | 100% |
| Diploid G1 | 82% |
| Diploid G2 | 6.72% |
| Diploid S | 11.28% |
| G2/G1 | 1.90 |
MultiCycle (with singlet discrimination)
| Diploid | 100% |
| Diploid G1 | 79.77% |
| Diploid G2 | 4.57% |
| Diploid S | 15.65% |
| G2/G1 | 1.92% |
Common Cell Cycle Dyes Used at The Facility
Probes | Facility Instruments | Excitation laser line | Emission Filter [nm] |
Propidium Iodide | BD LSR II | 488 nm | 695/40 |
Hoechst | BD LSR II | 407 nm | 450/50 |
7 Aminoactinomycin D (7AAD) | BD LSR II | 488 nm | 695/40 |
DAPI | BD LSR II | 407 nm | 450/50 |
DRAQ5 | BD LSR II | 640 nm | 660 /20 |
Links
Fluorescence Spectra of Afformentioned Dyes
Vendors
Thermo Fisher
Sigma
Vybrant DyeCycle Stains
Software
FCS Express with MultiCycle
Protocols
Determining Cell Cycle Stages by CMtO
Mitochondrial growth and DNA synthesis occur in the absence of nuclear
DNA replication in fission yeast by Sazer and Sherwood
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.
Apoptosis Analysis
At a late stage in the apoptotic cascade, a specific endonuclease breaks the DNA at the linkers between the nucleosomes resulting in a large number of small fragments (oligomers of about 180 bp). The DNA strand breaks may be detected by observation of a sub-G1 peak in the DNA histogram.
Annexin V
In normal viable cells, phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. In apoptotic cells however, PS is translocated from the inner to the outer leaflet of the plasma membrane thus exposing PS to the external cellular environment.
Changes in Mitochondrial Membrane Potential
DiOC6 is a green-fluorescent cationic dye that accumulates in active mitochondria and is useful in following changes in the membrane potential of the mitochondria that occur during programmed cell death.
Caspase Assay
In the early stages of apoptosis, cysteine-aspartic acid specific proteases called caspases are activated. The activation can occur through intrinsic and/or extrinsic pathways. Caspases destroy essential cellular proteins leading to controlled cell death.
Links
Fluorescence Spectra of Afformentioned Dyes
Annexin V – Alexa 488 conjugate
Vendors
Molecular Probes
Sigma
Software
FCS Express
Further reading
Flow Cytometry of Apoptosis (different methods: Rhodamine 123, Zenon Technology, FLICAs, Annexin V, PI, DAPI, TUNEL Assay etc.)
Protocol for staining dead cells with PI from Cancer Research Lab in UK
Protocol for PI staining (also cell cycle for adherent cells) from Arizona Research Labs
Apoptosis Analysis Guide from abcam
BDBiosciences Apoptosis Detection Kit
ThermoFisher Apoptosis Detection Kit
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.
Immunophenotyping
Direct staining
Fluorescent probe attached to primary antibody:
Protocols on the WEB
Direct Immunofluorescence Staining for CMtO from AbDSerotech
Direct Flow Staining Protocol from Abcam
Indirect staining
Fluorescent probe attached to a secondary antibody:
Protocols on the WEB
Indirect Immunofluorescence Staining for CMtO from AbDSerotech
Indirect Flow Staining Protocol from Abcam
Avidin-Biotin Method
Biotynylated antibody can bind many avidins (streptavidins) – fluorochromes complex to improve sensitivity of assay:
Zenon® Labeling Technology
Zenon technology is based on the complexation of primary antibodies with dye– or enzyme–labeled Fab fragments of secondary antibodies directed against their Fc regions.
Picture obtained from invitrogen webpage about Zenon® Labeling Technology
General Staining techniques
Surface Staining Protocol from University of Utah Flow Cytometry Facility
Vendors
Biocampare Antibodies
Biolegend
Thermo Fisher Antibodies
BD Biosciences Multicolor
EMD Millipore Antibodies
affymetryx eBioscience Immunology
Abcam
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.
Intracellular Antigen
Effective fixation and permeabilization are the most important parts in intracellular antigen staining as shown as the right peak on the bottom picture:
There are different methods for fixation and permeabilization used. The most important is to access the internal epitope of interest without disturbing the morphology of the cell.
Different methods of fixation and permeabilization available at Abcam website:
- Formaldehyde followed by detergent
Detergents:
- Triton or NP-40 (can partially dissolve the nuclear membrane)
- Tween 20, Saponin and Leucoperm (mild membrane solubilizers)
- Formaldehyde followed by methanol
- Methanol followed by detergent
- Acetone fixation and permeabilization
Pre-staining procedure:
- Add 100 µl of fixative. Incubate for 10 minutes at required temperature.
- Add 100µl detergent based permeabilizing agent and incubate in the dark at room at room temperature for 15 minutes.
- Wash the cells by adding 2ml of PBS (containing 0.1% triton or other permeabilizing detergent), centrifuge at 300g (2000 rpm) for 5 minutes, discard supernatant and re-suspend the pellet in the volume remaining.
- Follow antibody-staining procedure.
Links
CMtO Intracellular Staining Guide
Intracellular Antigen Staining
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.
Cell Counting
See also CELL PREPARATION
Cytometry and Microscopy to Omics can be used in the realm of cell counting where differentiation of multiple populations is necessary, e.g. in blood samples. Flow cytometric cell counting utilizes a sample with a known concentration of fluorescent beads. The sample is run through a flow cytometer and set to stop after a predetermined number of beads are analyzed. Because the concentration of beads is known, the volume of sample analyzed can be easily computed. The number of cellular events can then be easily counted and the concentration determined. Different populations within a sample can be differentiated by scatter properties or fluorescent staining.
The concentration of cells can be found by the following equation:
In the example above the bead concentration was known to be 2.0∙10^5 beads/mL so the concentration of bacteria can be found by:
Several companies offer tubes with known quantities of beads or solutions of known bead concentration. AccuCheck Counting Beads and ThermoFisher CountBrightTM absolute counting beads each contain known concentrations of beads. When using these products add a known volume of product to a known volume of sample, this will allow easy calculation of final bead concentration. BD TruCOUNT tubes contain a known quantity of beads. With these tubes add a known volume of sample directly to the tube, then calculate the bead concentration.
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.
Cytometric Bead Array
Picture obtained from BD Cytometric Bead Array – Multiplexed Bead-Based Immunoassays
If you are looking for a good alternative to enzyme-linked immunosorbent assays to efficiently and simultaneously measure different panels of analytes (chemokines, cytokines, phosphoproteins, growth factors, human immunoglobulins etc.) in a single microplate well, then multiplex bead assay is your choice.
Our Main Facility is equipped with a Bio-Rad Luminex Cytometric Bead Analyzer, which can perform up to 40 assays (each bead is infused with fluorescent dyes in different ratios allowing for emission of different amounts of photons depending upon the amount of binding) in a single well, increasing assay flexibility as it can be used to perform immunoassays, nucleic acid research, enzymatic research, and receptor-ligand studies.
Picture obtained from BIO-RAD – Multiplex Immunoassays
Multiple kits are available on the market, preconfigured or configurable kits (build your own multiplexes).
BD CBA Flex Sets – BD Biosciences
BD CBA Kits – BD Biosciences
Immunoassays and Multiplex kits – EMD Millipore
FlowCytomix Multiplex kits – affymetrix eBioscience
Human cytokine Multiplex kits – Life Technologies
Mouse cytokine Multiplex kits – Life Technologies
Bio-Plex Suspension Array System – BIO-RAD Luminex Assays and Luminex High Performance Assays – simultaneous detection and quantification of multiple target analytes in qualified complex sample types. Designed to provide accurate, reproducible results for every target analyte.
A paper to read if you want to know more:
Bead-Based Multianalyte Flow Immunoassays – The Cytometric Bead Array System
Scheduling
Please contact us to schedule an appointment or to discuss a future project: kjans01s@illinois.edu, (217) 300.5904.
Functional Assays
Cell Proliferation
Surface Antigen Quantification
Quantitative Flow Cytometry (QCFM) can be used to analyze large amounts of cells for multiple antigens and for quantifying the number of binding sites per cell. Linear function represents the relationship between mean fluorescence intensity (MFI) of cells that were labeled with a fluorochrome that was attached to a ligand or monoclonal antibody (mAb) and mean number of receptors/cells or mean number of mAb binding sites/cell. Special calibrated beads are used to generate a standard curve that converts MFI into molecular equivalents of soluble fluorochrome (MESF) by binding a quantifiable amount of antibody (Antibody Binding Capacity – ABC). The example below shows quantification of anti-Human EGFR on the cell surface of Human squamous carcinoma cell line SQCCY1 with DAKO QIFFIT® – picture obtained from U.T.M.D. Anderson Cancer Center Science Park – Research Division website. Different populations of beads showed in the picture bind calibrated amounts of CD5-FITC antibody. This kit measures immunofluorescence indirectly.
You can use series of five fluorescent microsphere populations labeled with varying amounts of FITC or other known fluorophores like Alexa Fluor® 488, Alexa Fluor® 647, APC, Cy™5, PE-Cy™5, R-PE to measure expression levels of cells. Fluorescence intensity is quantified through direct comparison of fluorescence measurements from pure fluorochromes with those from microspheres surface-labeled with the same fluorochrome.
FREE analysis program (QuickCal® v.2.3) is provided with each kit to help you determine expression levels of cells, and also to evaluate instrument linearity and detection threshold. The analysis program can be obtained HERE and used by entering the Access Number that was provided with your standards.
Links And Useful Information
- Article on comparison of the three different techniques for direct and indirect immunofluorescence.
- Bangs Laboratories beads for MESF analysis
- How to use FCS Express to create channel calibration on your own data (video)
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.
Cell Preparation for Analysis and Sorting in CMtO
Depending upon your type of cells you need to transfer them from a medium and perform cell count and viability tests.
- Harvest the cells (if obtaining from tissue), decant (if grown in the flask) and centrifuge them for 4-5 minutes (300-400xg) at 4°C and discard the supernatant.
- Resuspend the pellet in PBS or serum-free medium.
- Centrifuge for 4-5 minutes (300-400xg)
- Resuspend the pellet and perform cell count and viability analysis.
You can use hemocytometers for cell viability and cell count. You can use our automatic cell counter in the 231 ERML.
- Count the cells in squares 1,2,3,and 4.
- Record, average and multiply by dilution factor (if cells were not diluted, then it is 10^4/mL).
Example:
1-60 cells; 2-66cells; 3-58 cells; 4-68 cells; Average – 63 cells
63 cells * 10^4/mL = your concentration
In order to determine cell viability you need to stain your cells either with trypan blue (positive test will illuminate blue) or erythrosin B (positive test will illuminate red). In flow cytometry, you can use fluorescent dye – Propidium Iodide to determine cell viability.
- Mix your cell suspension 1:1 with erythrosin B solution in PBS or 0.4% trypan blue solution in PBS.
- Load the cells with erythrosin B solution to hemocytometer, but remember to incubate the cells with trypan blue for 2-5 minutes before loading
- Nonviable cells will be stained red (erythrosin B) or dark blue (trypan blue). Cell viability is expressed as a percentage of unstained cells divided by the total number of cells.
We offer users cell sorting in two different ways:
- Tubes (up to 4-way)
- Well plates format.
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.
Need help designing your panel?
Here are some tools that can help you design your panel with minimal spillover and as per our instrument configurations:
1. Have a look at the fluorochrome reference chart to make sure you have fluorochromes that are compatible with our instruments configuration
2. Use the BD Biosciences spectrum viewer to look at the spectrum overlap between your fluorochromes
3. Have a look at the below image to help you assign your fluorochromes to the appropriate populations
You can find more information on how to select your multicolor fluorochromes here
4. Contact the CMtO staff if you have any questions at CMtO-Core@mx.uillinois.edu
BIOSAFETY RULES FOR CYTOMETRY AND MICROSCOPY TO OMICS USERS
All researchers and their staff must know the potential biohazards associated with their work. Cytometry and Microscopy to Omics is suitable as a BSL-1 (Biological Safety Level 1) laboratory in any facility, and as BSL-2 in 1230 DCL, 348 Burril Hall, 377 Morrill Hall, 264 RAL and in 231C ERML. BSL-3 work is never permitted. Notify Facility staff if you are planning work at a higher level than BSL-1.
All users must follow these guidelines:
— NEVER bring food or drink in the laboratory.
— NEVER bring any radioactive materials into any of our labs.
— Self-service users are responsible for removing any trash from the bench areas, as well as cleaning up any spills.
— Facility staff must be notified if the spill is hazardous. If you need help they will assist you in getting it cleaned up.
— All sample-handling procedures must be done carefully to avoid generating aerosols.
— The use of a lab coat and gloves are required for BSL-2 work. Change gloves immediately if they become contaminated.
— Do not throw any laboratory materials into the regular trash. They may be discarded in our orange biohazard bags, but they must be decontaminated first. If you have biohazardous trash please dispose of it properly in your own lab. Samples containing hazardous chemicals (for example, propidium iodide) also must be taken back to your lab and held for pickup by the Division of Research Safety (http://www.drs.illinois.edu/).
— Self-service users must follow the established protocols for decontamination/rinsing of the sample lines within the instruments and must properly dispose of all waste materials after every analysis session.
— Any questions or comments please contact Cytometry and Microscopy to Omics at Cmto-core@mx.uillinois.edu, 217.300.5904
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.
