The Centre for Flow Cytometry & Scanning Microscopy (CCSM)

To use instruments in the Centre for Flow Cytometry and Scanning Microscopy (CCSM), you must provide a cost centre and undergo training on the specific instrument.

To schedule time in the CCSM, please book via Book My Lab.

To use this site you must be a registered member of the Centre for Cytometry and Scanning Microscopy (CCSM).

To become a registered member, navigate to the above link and click on the ‘Join Us’ button. Complete the required fields and submit. Please allow one business day for approval of your new account.

Please see the instructions below for more information.

Scheduling unassisted appointments

Adding appointments to the schedule

• Go the ‘calendar’ tab to view today’s appointment schedule. The DAY VIEW Box at the top of the schedule will have a red box around it.
• To add a new booking, click on the schedule for the correct instrument and drag to the right for the length of time you wish to reserve.
• Move your mouse to the small black box that appears over your request and click the blue book button.
• Fill all fields in with as much detail as possible and click the ‘save’ button at the bottom of the ‘add new record’ dialogue window.

Deleting/changing appointments

• Go to your reservation and click ‘edit’.
• There is a ‘cancel’ button at the bottom right side of the window.
• OR, go the ‘schedules’ tab menu and view the list of appointments you have made and cancel the appointment here.

Scheduling and cancellation rules

• Bookings that total more than 16 hours in a week must be approved by CCSM staff.
• You have up until 12 hours before an appointment to cancel your appointment with no penalties.
• Cancelling an appointment with less than 12 hours notice the appointment will result in full charges for the unused time on your supervisor’s account.
• If an appointment is missed, the session will be charged in full.
• If instruments are found to be left on overnight a $300.00 penalty fee will be applied to your account.

Editing appointments (analyzers and microscopes)

• Appointments deleted within 12 hours of initial start time will be charged without exception.
• If adjustments to timing or instruments are necessary, edit the appointment to move the appointment to a new time or instrument. It is important that the time on accurately reflects the actual time spend on the instrument.

Requesting appointments with operators – Assisted-acquisition

Data analysis, consultations and training requests:

• Requested by email only.
• Operator schedules are locked and are not bookable by users.

Assisted acquisition

• Book the instrument on which you would like work done.
• You must choose a service, i.e. ‘Assisted-Acquisition.’
• Select an operator (Paul Oleynik) then fill in as many details about your appointment as necessary.
• Please indicate the biosafety levels of the samples you would like sorted so operators can take the necessary personal protective measures.
• Save the booking and your name will appear on both the operator’s and instrument schedule (accounts are charged only for the operator’s schedule).
• Acquisition/analysis appointments do not start before 9:30 a.m.
• All assisted appointments must be finished before 5 p.m.
• If requesting an appointment with less than 24 hours notice, please notify the operator by email as we are not directly updated re-schedule changes.

Cancelling operator assisted appointments

• Cancel the appointment on the instrument schedule; the corresponding appointment on the operator’s schedule will also disappear.
• Appointments with the operator should be cancelled with 24 hours (or more) notice to avoid fees for the unused time.
• Appointments that are cancelled with 24 (or less) hours notice will be charged in full.
• Please let the operator know, by email, that you have cancelled your appointment, especially if cancelling with short notice.

Alexa fluor dyes

Although secondary labeling can significantly enhance a fluorescent signal, a direct conjugate is preferred because it creates minimal background fluorescence and less nonspecific binding. The Alexa dyes are extremely bright and are more photostable than comparable dyes in many cases.

Here is a handy comparison chart:

Cell surface staining protocol

FACS buffer (FB) recipe:

1x HBSS with 2% FBS and 12 mM HEPES. Optional 0.1% sodium azide to prevent microbial growth and 1 mM EDTA to prevent clumping.

Protocol:

1. Harvest, count and wash your filtered single cell suspension.
2. Aliquot 0.1–0.5 x 106 cells into conical polystyrene 5 mL tubes or 96-well round or V-bottom plates for staining.
3. Add FB and spin at 1100 rpm for five minutes.
4. Prepare appropriate dilutions of antibodies in FB and add 50-100 uL per sample.
5. Stain in the dark on ice or at 4°C for 30 minutes.
6. Wash twice with FB.
7. Resuspend in 500 uL FB with a live dead marker such as PI, DAPI or 7-AAD.
8. In order to prevent clogging of the flow cytometer, re-filter the cell suspension through a 70 μm cell strainer if clumps can still be observed.

Notes:

1. Cells expressing high levels of Fc receptors will contribute to non-specific binding. Fc blocking is recommended after step 3 and prior to staining with specific antibodies. Fc block should be used according to the manufacturer’s directions.
2. Appropriate controls need to be prepared and run prior to acquiring your fully stained samples. For multi-colour experiments, single stained controls for each fluorophore are mandatory in order to set up compensation.
3. In order to accurately gate cells when using a multi-colour panel, fluorescence minus one (FMO) controls are ideal. Fluorescence minus one controls allows you to identify appropriate gating strategies in the context of data spread due to numerous fluorochomes. An FMO contains all the fluorophores except the one of interest. For example, FITC FMO control contains all fluorophores in the stain except FITC.

Fixatives

For DNA samples, ice cold 70% ethanol added quickly to 100 uL cell suspension with rapid vortexing.

For antigen-DNA combinations, standard fixative is ice cold 70% ethanol added quickly with low speed vortexing after antibody staining and prior to DNA stains.

Please note that some antigens will not tolerate ethanol. Test the effect with FACS first before continuing. You may need to use a low paraformalydhyde concentration of 0.5% instead of ethanol. The CVs of your DNA peaks will be wider.

For neutralization of human samples or unknown biohazards, use 1% paraformaldehyde (always prepared fresh in PBS, pH 7.4) for 10 minutes.

For long-term storage (e.g. one to two weeks) of fluorochrome-stained surface antigens, use 0.5% paraformaldehyde.

Please note that the longer you store cells in paraformaldehyde, the more cellular autofluorescence you will observe.

Fluorochrome table

Download the common fluorochromes in flow cytometry table (PDF)

The table has characteristics for fluorochromes used in flow cytometry, within groups, roughly in order of excitation wavelength.

Colours are only a rough approximation.

Immunofluorescence staining protocol

1. Cell preparation
   • Adherent cells:
     • Grow desired cells on glass coverslips (preferably thickness #1) in six-, 12- or 24-well plates.
       Note: For analysis such as live imaging, grow cells on round glass coverslips.
   • Suspension cells:
     • Coat coverslips with excess polylysine (0.01% solution) for 10 minutes at room temperature. Aspirate the excess and let the coverslips dry. Rinse coverslips with water and place them in six-, 12- or 24-well plates.
     • Wash cells with PBS and resuspend. Add the resuspended cells onto the polylysine-coated coverslips and let them settle for 30–60 minutes. Aspirate the excess followed by a gentle rinse with PBS.
2. Fixation
   • For adherent cells, wash the coverslips with PBS at least three times to completely remove the media.
   • Replace PBS with one of these fixation solutions below.
   • There are different methods for fixing cells:
     • 4% paraformaldehyde (PFA) in PBS for 30 minutes or more at room temperature
       Note: This is a common method used for cell fixation. The 4% PFA can be made by diluting 16% PFA stock. It is recommended to prepare 4% PFA fresh.
     • 100% chilled methanol at -20°C for 10 minutes
       Note: This method fixes cells rapidly, thus useful for cytoskeleton staining, but can cause shrinkage of cells. If cells are fixed by this method, the permeabilization step can be skipped. In addition, this method cannot be used for fluorescent conjugated proteins (e.g. GFP), transfected cells and phalloidin staining.
   • After fixation, wash the coverslips with PBS at least three times to remove the fixation buffer.
3. Permeabilization
   • Permeabilize with 0.1–0.05% Triton X-100 in PBS (containing 100 mM glycine) for 20 minutes at room temperature.
     Note: Permeabilization is used when staining for internal antigens.
   • After permeabilization rinse the coverslips with PBS at least three times.
4. Blocking
   • There are different blocking solutions that can be used. Please check with the primary antibody information sheet for specifications.
     • Block with 2–5% animal serum, BSA or milk diluted in PBS for one hour at room temperature.
5. Incubating with primary antibody
   • Incubate the coverslips with diluted primary antibody in 1% blocking buffer for one hour at room temperature.
     Note: In general primary antibodies are diluted at (1:100–1:1,000). For specifications on dilution please check with the antibody’s information sheet. In order to conserve antibodies, the coverslips can be picked up by a tweezer and flipped onto a drop of antibody solution (75–100 μL) that can be placed on a piece of parafilm.
   • Rinse the coverslips with PBS at least three times.
6. Incubating with secondary antibody
   • Incubate the coverslips with diluted secondary antibody in 1% blocking buffer for one hour at room temperature.
     Note: In general, secondary antibodies are diluted at (1:1,000-1:1,500). Secondary antibodies from Jackson are the preferred choice. It is recommended to cover the plates with foil or place them in dark in order to limit bleaching of samples from this stage of the procedure.
   • Rinse the coverslips with PBS at least three times.
     Note: If cells will be stained with DAPI, the coverslips need to be rinsed with water at this stage before proceeding with DAPI staining. In general, for DAPI staining, incubate the coverslips with 1:10,000 diluted DAPI for 10 minutes then rinse with water.
7. Mounting
   • There are different mounting media that can be used: DAKO, Mowiol or PBS:Glycerol (1:1) followed by clear nail polish to seal coverslips.
     • Flip the coverslip onto a drop of mounting media placed on a slide.
       Note: Remove any air bubbles trapped between the coverslip and slide.
   • Leave the slides to dry overnight.

Immunophenotyping tip sheet

Understanding Fc blocking

Intact antibodies will bind to any cell that has an unoccupied Fc receptor. Only thymocytes and erythrocytes are void of Fc receptors on their cell surface. It is important to use an Fc block to bind up the Fc sites prior to incubation with antibodies. CD16/32 specifically binds to Fc receptors and can actually be used in conjunction with a second antibody to identify Fc receptor-carrying cells.

Understanding dead cells

Dead cells have the ability to pick up free-floating fluorescence dyes. By using a dead cell marker such as propidium Iodide, 7-AAD or DAPI, cells that have a permeabilized nuclear membrane can absorb these dyes, which bind to nucleic acids. These dyes fluoresce and can be subsequently excluded from data acquisition via live cell gating.

Understanding binding

There are two kinds of binding: specific and non-specific. Specific activity includes Fab to epitope and Fc to Fc Receptor. Non-specific is an across-the-board low-affinity stain that is non-saturable. Antibodies do not bind due to over-conjugation, non-purification, binding site degradation or aggregation.

Understanding antibody dilutions

Most antibodies purchased from companies arrive with suggested working dilutions. Perform a titration on these antibodies to ensure they work and to determine the stain index. This ensures you are using the optimal dilution to obtain the best resolution between the positive and negative fraction.

Sorting and aggregates

Aggregates or clumps can severely affect the integrity of your sort no matter which instrument you work with.

Clumps cause clogs, which block or disturb the laminar flow process through the nozzle. Such disturbance usually results in redirection of the central core stream or spraying of the side streams, and usually affects the purity of the sort.

While we can manually manipulate the instrument to counteract some of the hazards of aggregates, three bench-based steps can help:

1. Use PBS or HBSS without calcium and magnesium as the base of all the solutions you need in prepping cells for sorting. In some cases 1 mM EDTA is also recommended.
2. Keep protein in your sort buffer to 0.5% (FCS or BSA). Collection vessels can have unlimited protein to keep cells happy once they’ve been sorted.
3. Filter samples immediately prior to sorting by passing them through 50-70 um nylon mesh.

For primary tissue that has been homogenized or dissociated:

Cells need to be passed through a sieve to remove obvious chunks prior to staining but then, they should be filtered as described above just before the sort. The standard sorting nozzle size is 70 um.

For cultured cells, especially cells that don’t like to be removed from a matrix or that tend to adhere:

In most cases, cells are sorted at slightly lower pressures and in more dilute concentrations. For very large cell types, standard sort nozzle size is increased to 100 um and the pressures and speeds are reduced even further. Regardless of the size of the cell, it must be filtered prior to sorting if it comes out of a culture dish.

LSR II configuration guide

Common configurations

Special configurations

LSR II optical setup sheet

Download PDF version

Propidium iodide DNA staining protocol

Prepare in advance

1. RNASE solution: 2 mg/mL RNAse A (Bovine Pancreas Type II, Sigma) in HBSS. Divide into 500 ul aliquots and freeze.
2. Propidium iodide: 0.1 mg/mL PI (Sigma) in HBSS with 0.6% NP-40.
3. 1 x PBS with 2% FBS.

Procedure

1. Prepare cell suspensions.
2. Adjust concentration to 2 x 106 cells/mL, transfer to conical tubes and centrifuge at 1100 rpm (300 g) for five minutes.
3. Resuspend cells in 500uL PBS. Fix cells by adding 1 mL ice cold 70% ethanol dropwise. Vortex gently while adding ethanol. Fix on ice for at least 2 hours.
4. Centrifuge for five minutes and aspirate the supernatant. Wash once with PBS (may have 2% FBS).
5. Resuspend pellet in 500 uL 0.1 mg/mL propidium iodide (Sigma) with 0.6% NP-40. Gently vortex to mix.
6. Add 500 uL of 2 mg/ml RNAse solution to cells. Gently vortex and incubate cells in dark at room temperature for 30 minutes.
7. Filter and keep cold and in dark until analysis. Cells are analyzed in this DNA staining solution.

Sorting and aggregates tip sheet

Aggregates or clumps can severely affect the integrity of your sort no matter which instrument you work with.

Clumps cause clogs, which block or disturb the laminar flow process through the nozzle. Such disturbance usually results in redirection of the central core stream or spraying of the side streams, which affects the purity of the sort.

While we can manually manipulate the instrument to counteract some of the hazards of aggregates, three bench-based steps can help:

1. Use PBS or HBSS without calcium and magnesium as the base of all the solutions you need in preparing cells for sorting. In some cases 1 mM EDTA is also recommended.
2. Keep the protein concentration in your sort buffer to 0.5% (FCS or BSA). Collection vessels can have unlimited protein to keep cells happy once they’ve been sorted.
3. Filter samples immediately prior to sorting by passing them through 50–70 μm nylon mesh.

Below is a list of relevant published research from SRI and external scientists made possible via the Centre for Flow Cytometry and Scanning Microscopy instruments and personnel since 2010:

Allan DS, Kirkham CL, Aguilar OA, Qu LC, Chen P, Fine JH, Serra P, Awong G, Gommerman JL, Zúñiga-Pflücker JC, Carlyle JR. An in vitro model of innate lymphoid cell function and differentiation. Mucosal Immunol. 2015 Mar;8(2):340–51

Stanojcic M, Chen P, Harrison RA, Wang V, Antonyshyn J, Zúñiga-Pflücker JC, Jeschke MG. Leukocyte infiltration and activation of the NLRP3 inflammasome in white adipose tissue following thermal injury. Crit Care Med. 2014 Jun;42(6):1357–64

Zarin P, Wong GW, Mohtashami M, Wiest DL, Zúñiga-Pflücker JC. Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals. Proc Natl Acad Sci U S A. 2014 Apr 15;111(15):5658–63.

Benveniste P, Serra P, Dervovic D, Herer E, Knowles G, Mohtashami M, Zúñiga-Pflücker JC. Notch signals are required for in vitro but not in vivo maintenance of human hematopoietic stem cells and delay the appearance of multipotent progenitors. Blood. 2014 Feb 20;123(8):1167–77.

Fang L, Deng Z, Shatseva T, Yang J, Peng C, Du WW, Yee AJ, Ang LC, He C, Shan SW, Yang BB. MicroRNA miR-93 promotes tumor growth and angiogenesis by targeting integrin-β8.Oncogene. 2011 Feb 17;30(7):806–21.

Lee DY, Jeyapalan Z, Fang L, Yang J, Zhang Y, Yee AY, Li M, Du WW, Shatseva T, Yang BB. Expression of versican 3′-untranslated region modulates endogenous microRNA functions. PLoS One. 2010 Oct 25;5(10):e13599.

Du WW, Yang BB, Shatseva TA, Yang BL, Deng Z, Shan SW, Lee DY, Seth A, Yee AJ. Versican G3 promotes mouse mammary tumor cell growth, migration, and metastasis by influencing EGF receptor signaling. PLoS One. 2010 Nov 5;5(11):e13828.

Jeyapalan Z, Deng Z, Shatseva T, Fang L, He C, Yang BB. Expression of CD44 3′-untranslated region regulates endogenous microRNA functions in tumorigenesis and angiogenesis. Nucleic Acids Res. 2011 Apr;39(8):3026–41.

Shatseva T, Lee DY, Deng Z, Yang BB. MicroRNA miR-199a-3p regulates cell proliferation and survival by targeting caveolin-2.J Cell Sci. 2011 Aug 15;124(Pt 16):2826–36.

Allan DS, Rybalov B, Awong G, Zúñiga-Pflücker JC, Kopcow HD, Carlyle JR, Strominger JL. TGF-β affects development and differentiation of human natural killer cell subsets. Eur J Immunol. 2010 Aug;40(8):2289–95.

Awong G, Herer E, La Motte-Mohs RN, Zúñiga-Pflücker JC. Human CD8 T cells generated in vitro from hematopoietic stem cells are functionally mature. BMC Immunol. 2011 Mar 23;12:22.

Mohtashami M, Shah DK, Nakase H, Kianizad K, Petrie HT, Zúñiga-Pflücker JC. Direct comparison of Dll1- and Dll4-mediated Notch activation levels shows differential lymphomyeloid lineage commitment outcomes.J Immunol. 2010 Jul 15;185(2):867–76. Erratum in: J Immunol. 2010 Sep 15;185(6):3777–8.

Wang D, Claus CL, Rajkumar P, Braunstein M, Moore AJ, Sigvardsson M, Anderson MK. Context-dependent regulation of hematopoietic lineage choice by HEBAlt.J Immunol. 2010 Oct 1;185(7):4109–17.

Braunstein M, Anderson MK. Developmental progression of fetal HEB(-/-) precursors to the pre-T-cell stage is restored by HEBAlt.Eur J Immunol. 2010 Nov;40(11):3173–82.

Braunstein M, Anderson MK. Developmental progression of fetal HEB(-/-) precursors to the pre-T-cell stage is restored by HEBAlt.Eur J Immunol. 2010 Nov;40(11):3173–82.

Braunstein M, Rajkumar P, Claus CL, Vaccarelli G, Moore AJ, Wang D, Anderson MK. HEBAlt enhances the T-cell potential of fetal myeloid-biased precursors.Int Immunol. 2010 Dec;22(12):963–72.

Braunstein M, Anderson MK. HEB-deficient T-cell precursors lose T-cell potential and adopt an alternative pathway of differentiation.Mol Cell Biol. 2011 Mar;31(5):971–82. Epub 2010 Dec 28

Fine JH, Chen P, Mesci A, Allan DS, Gasser S, Raulet DH, Carlyle JR. Chemotherapy-induced genotoxic stress promotes sensitivity to natural killer cell cytotoxicity by enabling missing-self recognition. Cancer Res. 2010 Sep 15;70(18):7102–13.

Chen P, Mesci A, Carlyle JR. CELLISA: reporter cell-based immunization and screening of hybridomas specific for cell surface antigens. Methods Mol Biol. 2011;748:209–25.

Vaags AK, Gartley CJ, Halling KB, Dobson H, Zheng Y, Foltz WD, Dick AJ, Kruth SA, Hough MR. Migration of cells from the yolk sac to hematopoietic tissues after in utero transplantation of early and mid gestation canine fetuses. Transplantation. 2011 Apr 15;91(7):723–30.

Burgess A, Ayala-Grosso CA, Ganguly M, Jordão JF, Aubert I, Hynynen K. Targeted delivery of neural stem cells to the brain using MRI-guided focused ultrasound to disrupt the blood-brain barrier. PLoS One. 2011;6(11):e27877.

Jordão JF, Ayala-Grosso CA, Markham K, Huang Y, Chopra R, McLaurin J, Hynynen K, Aubert I. Antibodies targeted to the brain with image-guided focused ultrasound reduces amyloid-beta plaque load in the TgCRND8 mouse model of Alzheimer’s disease. PLoS One. 2010 May 11;5(5):e10549.

Tomic J, Lichty B, Spaner DE. Aberrant interferon-signaling is associated with aggressive chronic lymphocytic leukemia. Blood. 2011 Mar 3;117(9):2668–80.

Shi Y, Tomic J, Wen F, Shaha S, Bahlo A, Harrison R, Dennis JW, Williams R, Gross BJ, Walker S, Zuccolo J, Deans JP, Hart GW, Spaner DE. Aberrant O-GlcNAcylation characterizes chronic lymphocytic leukemia. Leukemia. 2010 Sep;24(9):1588–98.

Buckley KM, Rast JP. Characterizing immune receptors from new genome sequences. Methods Mol Biol. 2011;748:273–98.

Amoodi HA, Mick PT, Shipp DB, Friesen LM, Nedzelski JM, Chen JM, Lin VY. Results with cochlear implantation in adults with speech recognition scores exceeding current criteria. Otol Neurotol. 2012 Jan;33(1):6–12.

Silver K, Harrison RE. Measuring immune receptor mobility by fluorescence recovery after photobleaching.Methods Mol Biol. 2011;748:155–67.

Emmenegger U, Francia G, Chow A, Shaked Y, Kouri A, Man S, Kerbel RS. Tumors that acquire resistance to low-dose metronomic cyclophosphamide retain sensitivity to maximum tolerated dose cyclophosphamide. Neoplasia. 2011 Jan;13(1):40–8

Nguyen VP, Chen SH, Pizzuto K, Cantarutti A, Terminesi A, Mendonca C, Dumont DJ. 25-Hydroxycholesterol exerts both a cox-2-dependent transient proliferative effect and cox-2-independent cytotoxic effect on bovine endothelial cells in a time- and cell-type-dependent manner. J Angiogenes Res. 2010 Nov 11;2:24.

Nguyen VP, Hanna G, Rodrigues N, Pizzuto K, Yang E, Van Slyke P, Kim H, Chen SH, Dumont DJ. Differential proteomic analysis of lymphatic, venous, and arterial endothelial cells extracted from bovine mesenteric vessels. Proteomics. 2010 Apr;10(8):1658–72.

Zittermann SI, Capurro MI, Shi W, Filmus J. Soluble glypican 3 inhibits the growth of hepatocellular carcinoma in vitro and in vivo. Int J Cancer. 2010 Mar 15;126(6):1291–301.

Li F, Shi W, Capurro M, Filmus J. Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling. J Cell Biol. 2011 Feb 21;192(4):691–704.

Ichim CV, Atkins HL, Iscove NN, Wells RA. Identification of a role for the nuclear receptor EAR-2 in the maintenance of clonogenic status within the leukemia cell hierarchy.Leukemia. 2011 Nov;25(11):1687–96.

The Centre for Flow Cytometry and Scanning Microscopy (CCSM) at the Sunnybrook Research Institute (SRI) offers cell analysis services. We provide sample acquisition and data analysis services, as well as training courses for flow cytometry. We also provide user assistance and training courses for imaging.

The facility also offers cell-sorting services. Please contact Paul Oleynik for more information.

For human cell sorting:

We are a BSL2 facility permitting cells from risk groups two and three. Please see the section on biosafety for a full explanation. We will not accept cells that could be infectious to humans via aerosol contamination. This includes any first-generation lenti- or adeno-viral constructs, as well as infectious primary human tissue.

Please fill out and attach a Human Cell Sorting Request Screen Form (.doc) when requesting sort appointments.

For nonhuman cell sorting:

Email Paul Oleynik and indicate the following:

• cell type
• fluorochromes you would like to use
• number of cells you are bringing.

We will contact you with an appointment.

For your appointment, remember to bring the following:

• collection tubes for your cells (plus two extra) containing media
• extra sort buffer in case we need to dilute.

Please review the protocol for sorting and aggregates.

Ideal cell concentrations (all samples must be filtered):

• Aria/Fusion: high-speed, 20–-30 million cells per ml
• Aria/Fusion: larger nozzle medium-speed, 10­–20 million per ml (based on cell type)
• Aria/Fusion: plate sorting, 5 million per ml

FACS training

Course details

FACS training is divided into the following components:

• Introduction to flow basics which covers components of a flow cytometer and basics of fluorescence absorption and emission
• Use of FACSCalibur instrumentation, data acquisition and analysis
• Hands-on practice session using beads or prepared sample.

We use FACSCalibur analyzers equipped with two lasers, Macintosh G4 and BD CellQuest Pro data acquisition and analysis software.

Training schedules depend on instrument availability. Courses generally occur twice a month and are filled on a first-come, first-serve basis. Each training session takes three to fourhours and is split up into one- or two-day sessions to allow for sample preparation. At the end of the course, users may need to take advantage of assisted FACS sessions to complete their learning.

Requirements

It is important that you understand your project objectives and the biology of your cells. You will not succeed in adopting flow as a technological tool if you don’t understand why you need to look at cells with flow cytometry.

Recommendations

We encourage you to bring the prepared cells or samples that you will most likely be working on for your project. Examples include GFP expression, DNA content or cell-surface immunofluorescence.

It is not advisable to register for the course if you do not have a sample or a FACS project ready (this includes antibodies and reagents).

LSRIILSR II and FACSCanto training

Course details

LSRIILSR II and FACSCanto training is divided into the following components:

• Advanced topics such as filter optimization, instrument configuration, bi-exponential display and digital data
• FACSDiva software
• Instrument setup and usage
• Quality control
• Acquisition and analysis

Training is available to users who are already experienced with flow via the FACSCalibur. If you do not have FACSCalibur training but absolutely require a panel using more than 4 fluorophores for your flow cytometry characterization, then training will be provided. Both the LSR II and FACSCanto use FACSDiva software, which is very different from CellQuest Pro. Digital flow cytometry requires due diligence in preparing the cytometer via single stained compensation controls and assigning appropriate PMT voltages. We focus on drawing your attention to improved quality control practices.

Some applications for which you can use the LSR II system include:

• multi-fluor assays (14 parameter)
• calcium flux assays
• phosphorylated protein assays
• H0342 side population assays
• Simultaneous CFP-GFP-YFP-dsREd measurements

Recommendations

An excellent understanding of your application or the biology of your cells is recommended. This will help you use the software effectively.