FAQ

Simple or multiple multiparametric immunostainings analysis by flow cytometry

Cell cycle and ploidy analysis by flow cytometry

Cell Viability by flow cytometry

Cytometric Bead Array (CBA) (BD Biosciences)

Cell sorting and cloning

Multiplexing

 

 

• What is the procedure to make a multiparametric immunostaining analysis by flow cytometry?

 

1. First of all, contact Sandra Ormenese, the head of the facility (04 366 49 38 or Sandra.Ormenese@ulg.ac.be or imaging.Giga@ulg.ac.be), or Raafat Stephan, the Flow Cytometry specialist (04 366 25 79 or Raafat.Stephan@ulg.ac.be) to discuss the type of analysis you are planing to do. They will advice you concerning the most adequate cytometer for your purpose and will take your reservation into account.2. If it is the first time you want to use this instrument, you will be asked to follow a training given by a member of the facility staff.  This training is required to ensure that the instrument is operated properly and with maximum efficacy.
3. Before any new experiment, be sure to have the Biosafety level 1 (FR/EN) form completed. This form has not to be filled for each analysis, but for any new experiment.  It is your responsibility to check that a form (under your name) describing samples and ongoing experiments is correctly completed and transmitted to Sandra Ormenese or to Raafat Stephan.

 

• Which florochrome do I have to use?

 

Due to an increasing number of reagents, there are many different possibilities for cell suspension immunostaining. To help you, you can use the BD spectrum guide.

Depending on the application and on the chosen fluorochromes, 5 cytometers (BD Biosciences) are available for analysis:FACSCanto II: for more than 3 fluorochromes (max 8 colors)FACSCalibur 1 laser (488nm): for GFP transfected cells  or staining with up to 3 colours using fluorochromes excited at 488nm (for example: FITC, PE, PerCP)FACSCalibur 2 lasers (488 et 633 nm): for up to 4 colours, including fluorochromes requiring a 633nm (for example: APC)FACSAria IIIu 4L SORP: max 10 colorsFACSVerse: walk-away system, max 8 colors

For further information concerning the excitation and emission spectra and the most appropriate combinations, refer to a spectrum viewer and have a look on our  general specifications of our available flow cytometers to be sure to use the most appropriate flow cytometer for reading your color panel. To design your panel, you can use the BD FACSelect Multicolor Panel Designer.

 

• At which concentration do I have to resuspend the cells?

 

For an accurate and rapid analysis, your cells should be resuspended at a concentration of 1 million cells/ml in a minimum volume of 300 µl.  Tubes provided by the facility have to be used. If necessary, contact Raafat Stephan or Sandra Ormenese.

• Do I need to fix the cells?

Samples that are potentially infected HAVE to be fixed. Generally, cells are fixed with 1-2% paraformaldehyde (PAF) prepared in PBS. 

• Do I need to add controls? You ALWAYS have to prepare a tube containing mock-labelled cells to determine their autofluorecence level.  If you have multiple staining, it is also important to prepare tubes containing cells labeled with one single fluorochrome (one tube for each fluorochrome). These samples are required to set up the parameters and electronic compensations. If necessary, a member of the facility staff will give you advice for the compensation settings. • Which software should I use to analyse my immunostaining data?
The softwares able to read the FCS files are the only ones that could be used for the data analysis.  Different softwares (FACSDiva, FACSuite, Cell Quest Pro and FlowJo) are available on a computer independent from the cytometers. The cytometers being used in priority for data acquisition, data analysis has to be done on this independent computer.  Graphs and tables generated by the analysis will be printed and could be saved as a PDF file. Raw data and/or analysed data have to be saved by the costumers on a USB flash drive. 

 

• Do I need to fix the cells?


Samples that are potentially infected HAVE to be fixed. Generally, cells are fixed with 1-2% paraformaldehyde (PAF) prepared in PBS.

• Do I need to add controls?


You ALWAYS have to prepare a tube containing mock-labelled cells to determine their autofluorecence level.  If you have multiple staining, it is also important to prepare tubes containing cells labeled with one single fluorochrome (one tube for each fluorochrome). These samples are required to set up the parameters and electronic compensations. If necessary, a member of the facility staff will give you advice for the compensation settings.

 

• Which software should I use to analyse my immunostaining data?


The softwares able to read the FCS files are the only ones that could be used for the data analysis.  Different softwares (FACSDiva, FACSuite, Cell Quest Pro and FlowJo) are available on a computer independent from the cytometers. The cytometers being used in priority for data acquisition, data analysis has to be done on this independent computer.  Graphs and tables generated by the analysis will be printed and could be saved as a PDF file. Raw data and/or analysed data have to be saved by the costumers on a USB flash drive.

 

• What is the procedure to proceed to cell cycle or ploidy analysis by flow cytometry?


1. First of all, contact Sandra Ormenese, the head of the facility (04 366 49 38 or Sandra.Ormenese@ulg.ac.be or Imaging.Giga@ulg.ac.be), or Raafat Stephan, the Flow Cytometry specialist (04 366 25 79 or Raafat.Stephan@ulg.ac.be) to discuss about the cell cycle analysis you are planing to do. They will take your reservation into account. 2. When the date of the analysis is scheduled, be sure to have the Biosafety level 1 ( FR/ EN) form completed. This form has not to be filled for each analysis, but has to be filled for any new experiment.  It is your responsibility to check that a form (under your name) describing the samples and the ongoing experiments has been correctly completed and given to Sandra Ormenese or Raafat Stephan.


• How do I have to prepare my samples?

 

Samples have to be labeled with fluorescent molecules that bind specifically and stoichiometrically to DNA, such as propidium iodide (PI), in order to obtain a linear relationship between cellular fluorescence intensity and DNA amount. When utilizing PI, which is not specific for DNA, it is essential to use a DNAse free RNAse preparation according to recommended concentration in procedure. The more simple solution is to use a kit commercialised for this kind of experiment (for example, Cycle TEST Plus DNA Reagent Kit -BD Biosciences). Don't forget to bring with you control cells whose ploidy is known (for example: human lymphocytes). With this monoparametric analysis, it is not possible to discriminate G2 or M cells, or to distinguish the proliferative, the quiescent or the transition stages for cells with the same DNA content. Moreover, it is sometimes impossible to distinguish on the histograms cellular populations with very similar DNA contents. All this explain the interest for multiparametric analysis. In the multiparametric approach, are used anti-5-bromodeoxyuridine (BrdUrd) monoclonal antibodies conjugated with FITC and the staining of total DNA content with PI or 7-amino-actinomycin D (7AAD).  The more simple solution is to use a kit commercialised for this kind of experiment (for example,  this BD Pharmingen BrDU Flow kits) With this multiparametric method it is possible to distinguish cells in early-, mid- and late- S phase (see Cell cycle analysis by flow cytometry: Principles and applications, Jaya and Ratinaud, Biol Cell (1993) 78, 15-25).

• Which software should I use to analyse my cell cycle or ploidy data?

 

The software to analyse the data for the monoparametric acquisition is ModFit (BD Biosciences). It is accessible on  a computer independent from the the cytometers. Graphs and tables could be printed and/or be saved as a PDF file. Raw data and/or analysed data will be saved by the costumers on a USB flash drive. Please, note that the G2:G1 ratio for the FACSCalibur 1 laser is 1.95 and that a guideline would be to aim for a CV of 4% for cell lines and 2% for primary cells.

 

• What is the procedure to proceed to cell viability by flow cytometry?


1. First of all, contact Sandra Ormenese, the head of the facility (04 366 49 38 or Sandra.Ormenese@ulg.ac.be or Imaging.Giga@ulg.ac.be), or Raafat Stephan, the Flow Cytometry specialist (04 366 25 79 or Raafat.Stephan@ulg.ac.be) to discuss about the cell viability analysis you are planing to do. They will take your reservation into account.
 
2. When the date of the analysis is scheduled, be sure to have the Biosafety level 1 ( FR/ EN) form completed. This form has not to be filled for each analysis, but has to be filled for any new experiment.  It is your responsibility to check that a form (under your name) describing the samples and the ongoing experiments has been correctly completed and given to Sandra Ormenese or Raafat Stephan.


• How do I have to prepare my samples?

 
Cell viability can easily be determined in flow cytometry by adding a DNA binding dye at relatively low concentration to a population of cells. You can use propidium iodide (PI) or 7-amino-actinomycin D (7-AAD) with caution as live cells will eventually take up these dyes if left to long. In addition, annexins are a family of calcium-dependent phospholipid-binding proteins, which bind to phosphatidylserine (PS) to identify apoptotic cells. In healthy cells, PS is predominantly located along the cytosolic side of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution in the phospholipid bilayer and translocates to the extracellular membrane, which is detectable with fluorescently labeled Annexin V. In early stages of apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI) and 7-AAD, therefore cells which display only Annexin V staining (PI/7-AAD negative) are in early stages of apoptosis. During late-stage apoptosis, loss of cell membrane integrity allows Annexin V binding to cytosolic PS, as well as cell uptake of PI and 7-AAD. Annexin V staining, paired with 7-AAD or PI is widely used to identify apoptotic stages by flow cytometry.
The more simple solution is to use a kit commercialised for this kind of experiment (for example the  Annexin V FITC apoptosis detection kit from ebioscience or the FITC Annexin V apoptosis detection kit from BDBiosciences (read also this application note if you you are planing to do your viability analysis on the FACSVerse)).
 

• Which software should I use to analyse my cell viability data?

 
To analyse your cell viability data, you can use the softwares for cytometry CellQuest Pro, FACSDiva, FACSuite or FlowJo. They are available on computers that are independant from the cytometers (in the data room). Computers piloting flow cytometers have to be used in prioritory for data acquisition and not for data analysis.

• What is the procedure to proceed to a Cytometric Bead Array analysis?


1. First of all, contact Sandra Ormenese, the head of the facility (04 366 49 38 or Sandra.Ormenese@ulg.ac.be or Imaging.Giga@ulg.ac.be), or Raafat Stephan, the Flow Cytometry specialist (04 366 25 79 or Raafat.Stephan@ulg.ac.be), to discuss the type of analysis, the number of samples to analyse and the flow cytometer you have to use : the FACSCanto II or the FACSVerse.

Pay attention please:If you are using a CBA kit on the FACSCanto II: please,  ead this guide.If you are using a CBA Flex Set on the FACSCanto II: please, follow this procedure for FACSDiva.If you are using a CBA kit or a CBA Flex Set on the FACSVerse: please, follow this procedure for FACSuite and read this application note. 2. If it is the first time you want to use the chosen flow cytometer, you will be asked to follow a training given by the staff of the facility.  This training which is specific for CBA analysis on FacsCanto II or on FACSVerse is required to assure that the instrument will be used properly and with the maximum efficacy.
3. When the date of the analysis is scheduled, be sure to have the Biosafety level 1 ( FR/ EN) form completed. This form has not to be filled for each analysis, but has to be filled for any new experiment.  It is your responsibility to check that a form (under your name) describing the samples and the ongoing experiments has been correctly completed and given to Sandra Ormenese or Raafat Stephan.
 

Which software should I use to analyse my CBA data?

 
The software to analyse the data for this application is FCap Array software ( see more) (BD Biosciences). It is accessible on  a computer independent from the the cytometers. Graphs and tables could be printed and/or be saved as a PDF file.
Raw data and/or analysed data will be saved by the costumers on a USB flash drive.
 

• How do I have to proceed to schedule a cell sorting or a cloning experiment ?


1. First of all, contact Sandra Ormenese, the head of the facility (04 366 49 38 or Sandra.Ormenese@ulg.ac.be or Imaging.Giga@ulg.ac.be), or Raafat Stephan, the Flow Cytometry specialist (04 366 25 79 or Raafat.Stephan@ulg.ac.be) to discuss about the sorting you plan to do. They will give you advice concerning the flow cytometer sorter that is the most adequate for your purpose and will take your reservation into account.2. When you come to perform your sorting, be sure to have the Biosafety level 2 ( FR/ EN) form completed. It is your responsibility to check that a form (under your name) describing the samples and the ongoing experiments has been correctly completed and given to Sandra Ormenese or Raafat Stephan.

What is necessary to prepare and to bring for the day of the cell sorting or cloning?

You must bring polypropylene tubes (12x75 mm) for adherent cells or polystyrene tubes (12X75 mm) for non-adherent cells. These tubes could be provided by Sandra Ormenese or Raafat Stephan on request.

a.     one "injection tube" with the cells to be sorted (see FAQs 3 et 6)
b.   the "collection tubes" with approximately 500 µl of buffer (sterile PBS or complete sterile culture medium with antibiotics -for exemple pen/strep- if the sorting has to be done under sterile conditions). If needed, the collection tubes should contain "lysis buffer" but pay attention that sorted cells are included in a PBS drop: the lysis buffer will therefore become diluted along the sorting.

In the case of a cloning, you must bring a 96-wells plate filled with complete sterile culture medium (with antibiotics). Don't forget to bring the form of Biosafety Level 2 (FR/EN) duly completed and signed.


• At which concentration and in which buffer do I have to resuspend the cells to be sorted?


In general, the ideal concentration is of 10.000.000 cells/ml in PBS (without serum and Ca/Mg2+ free) with possibly 1 to 2% of BSA, 10 mM EDTA and 5 to 25 mM HEPES. However, it depends on the cell type to be sorted. Indeed, certain cells tend to form aggregates at such concentrations and it will thus be worth in this case to better dilute them. If possible, bring 4 ml of cell suspension in one tube. 
 
• How many cell populations could be sorted?


In eppendorfs or FACS tubes (12 X 75 mm), one to four cell populations can be sorted simultaneously. In Falcon tubes (15 ml), it is maximum two-way sorting. Each population can be identified according to multiple parameters such as fluorescent staining or size. Live cells can be sorted on the basis of dead cell exclusion with low concentration of propidium iodide (PI) or 7-amino-actinomycin D (7-AAD). The sample chamber can be set to a specified temperature to keep the sample cold or warm (4, 20 or 37°C) and there is also a water recirculator system for refrigiration/heating of the sample collection tubes.


• How long lasts a cell sorting?


The duration of the sorting will depend on the quantity of sorted cells which you want to obtain and on the percentage of these cells in the initial sample. Attention: the maximum injection speed of the sample is 60 µl per minute: please, make the calculation to avoid to bring a too big quantity of suspension of cells to be sorted (i.e. it takes 66 minutes to inject 4 ml of cell suspension in the FACS).
If you want to sort a rare cellular population, it will take more time than if you want to sort cells already present at 30 or 40% in the initial suspension. If possible, rare events should be enriched through bulk method (i.e. magnetic sort). The duration of the sorting will also depend on the speed of injection of the sample to be sorted (max 10.000 cells per second), on the quality of the sample and the size of the cells. The formation of clogs due to the aggregation of the cells in the sample can also considerably lengthen the duration of the sorting. 

• How do I have to proceed to avoid a maximum of aggregates in the cells sample to be sorted?


For a good sorting, it is critical to keep the cell suspension as a monocellular suspension. Aggregates are often formed when one works with adherent  or activated cells, or when one starts from a sample containing a high percentage of dead or damaged cells: soluble DNA can make the intact cells sticking.EDTA (5mM) in the sample can help to avoid agglomerate.  Addition of DNAase II (10U/ml) can contribute to the elimination of soluble DNA. For adherent cells, some  reagents  like ACCUTASETM or trypsin inhibitors can be used. If these solutions are not sufficient, the sample can be filtered through a standard filter nylon Blutex (mesh with 40 µm) provided on request by the platform.


• Which are the advantages of the cell sorting by cytometry compared to other techniques of purification (for example: by magnetic beads)?


-    A higher purity
-    One can separate cell populations on the basis of their fluorescence intensity
-    A better separation of the populations using multiple stainings
-    The cells of interest have a greater capacity of recovery
-    Dead cells can be eliminated
-    One can also sort on the basis of intracellular staining (for examples DNA, expression of cytokines, GFP…)
-    Up to four cell populations can be sorted simultaneously
-    Cloning by flow cytometry is the only technique which makes it possible to know as a preliminary the phenotype of the isolated cell


 • What is the procedure to proceed a multiplexing analysis?


1. First of all, contact Sandra Ormenese, the head of the facility (04 366 49 38 or Sandra.Ormenese@ulg.ac.be or Imaging.Giga@ulg.ac.be), or Raafat Stephan, the Flow Cytometry specialist (04 366 25 79 or Raafat.Stephan@ulg.ac.be) to discuss about the multiplexing you are planing to do. They will take your reservation into account.

 

• How do I have to prepare my samples?

 
Bio-Plex assays are designed using microscopic beads, each with a different color code (spectral address) to permit discrimination of individual assays. Beads are dyed with different ratios of two fluorochromes and are thus classified into 100 unique bead regions (xMAP technology). Each bead lots are coupled to antibodies against a different target.
  • Add coupled beads to wells of the 96-well plate which is coming with the kit.
  • Wash
  • Add samples to wells containing coupled beads
  • Incubate 30 minutes
  • Wash
  • Add to wells the biotin-labeled detection  antibodies which are specific for secondary epitopes on each target.
  • Incubate 30 minutes
  • Wash
  • Add fluorescently labeled streptavidin reporter to wells
  • Incubate 10 minutes
  • Rinse
  • Resuspend in assay buffer
  • Read the assay on the Bio-Rad Luminex 100 reader: a red classification laser and a green reporter laser illuminate individual beads to identify each bead's spectral address and associated reporter signal.
  • Multiplex data are reported simulteanously.
 

• Which software should I use to analyse my multiplexing data?

 
The software to analyse the data for the multiplexing is the Bio-Plex Manager Software. Graphs and tables could be printed and/or be saved as a PDF file. Raw data and/or analysed data will be saved by the costumers on a USB flash drive. 

Le livre (in french) " CYCLE CELLULAIRE ET CYTOMETRIE EN FLUX" de Grunwald, Mayol et Ronot, aux éditions Lavoisier, est à votre disposition au sein de la plateforme. Demandez-le à  Sandra Ormenese ou  Raafat Stephan.


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