American Univesity of Beirut

Hours of operation: 8am- 5pm
Reservation link
Custodian: Dr. Samia Khoury and Dr. Layal El Hajjar
Access Level: Restricted
Location: DTS, 3rd floor, room 308-C

Equipment

Our BD FACSAria Special Order Research Product (SORP) cell sorter has 4 different laser excitation wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 640 nm (Red). It can detect FSC, SSC and up to 17 fluorochromes. The FACSAria SORP can perform 2 and 4 way sorting as well as automatic cell deposition for sorting onto TC plates or slides. The sorter is provided with three nozzles with varying sizes for sorting different cell types and sizes (70µm, 85µm and 100µm). Below is the list of filters available for each laser.

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Specifications of FACSAria SORP  ​ ​ ​ ​ ​ ​
​Optics  ​ ​ ​ ​ ​ ​
Fluorochrome	Excitation laserline	Ex.-Max	Em.-Max	LP_Filter	BP_Filter	Channel-PMT
FSC	488					Photodiode
SSC	488				488/10
FITC (Fluorescein)	488	495	519	505LP	530/30	488-F
Alexa Fluor 488	488	​	​	​	​	​
PE (R-Phycoerythrin)	488	496,564	576	550LP	575/26	488-E
PE-Texas Red	488	566	616	600LP	610/20	488-D
PI (Propidium Iodide)	488	536	617	​	​	​
PerCP	488	482	678	635LP	670/20	488-C
PerCP-Cy5.5	488	482	695	685LP	710/50	488-B
PE-Cy7	488	496,564	785	750LP	780/60	488-A
Pacific Blue	405	401	452	-	450/50	405-F
AmCyan	405	457	491	505LP	525/50	405-E
BD Horizon BV510	405	402	510	​	​	​
Qdot565	405	300	564	550LP	560/20	405-D
BD Horizon BV605	405	407	602	600LP	610/20	405-C
BD Horizon BV650	405	407	647	635LP	670/30	405-B
BD Horizon BV711	405	405	711	685LP	710/50	405-A
APC (Allophycocyanin)	640	651	660	-	660/20	640-C
Alexa 700	640	696	719	685LP	730/45	640-B
APC-Cy7	640	756	779	750LP	780/60	640-A
APC-H7	640	757	780	​	​	​
Hoechst Blue	355	352	455	-	450/50	355-B
DAPI	355	​	​	​	​	​
Hoechst Red	355			600LP	670/30	355-A

FACSAria SORP Cell Sorter Software

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The facility uses the BD FACS DIVA software which operates on a computer station in the facility itself. A second copy of FACS DIVA is placed on a station in the DTS building, 3rd floor, Room 308.

N.B.: The BD FACSAria SORP could be used for routine analysis/acquisitions in case no other flow cytometer is available for this purpose. We will accept samples with all dyes except Propidium Iodide

Reservation

• Reservations of the sorter via the online booking system are possible. You can access the reservation sheet under “Facility Reservation/ Faculty of Medicine/ BD FACSAria SORP Cell Sorter".
• Reservations are subject to approval by the operator and users are advised to verify their reservations 24 hours prior to the experiment in case of an emergency cancellation.
• Bookings should be cancelled as early as possible in case of a cancellation of an experiment.
• You should fill up the “FACS Aria Usage Form" required for sorting.
• The Facility manager must be notified before working with any hazardous substance and/or organism in the facility to take the proper precautions.

What to Bring with You

Samples should be brought in one of the followings:

• 5mL polystyrene 12x75mm tubes (BD Falcon, ref#: 352052 or 352054)
• 15mL conical centrifuge tubes (BD Falcon, ref#: 352096)
• 1mL microtubes (Bio-Rad, ref#: 223-9391)

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For sorted cells the following tubes or plates are acceptable:

• 5mL Polyropylene 12x75mm tubes (BD Falcon, ref#: 352063) (2-way and 4-way sorting)
• 15mL falcon tubes (2-way sorting)
• 1.5mL eppendorf tubes (4-way sorting)
• Multiwell plates

* Specially designed collection tube holders with ports for recirculating water are also available for 2-way 15mL tubes, 4-way 12x75mm tubes and 4-way 1.5mL Eppendorf tubes sorting.

** Polypropylene tubes should be used for sorting to allow optimal recovery of you cells since polystyrene tubes will cause the cells to adhere to the plastic.

The concentration of your cells

The starting number of your cells depends on the frequency of your target cells within the total cell population, the recovery and the percentage of the target sorted cells. For example, if you require 1x10⁵ cells of a particular type and this cell type is present at 10% of your total cells then you will need to sort at least 1x10⁶ cells. If it is only present at 1% then a least 1x10⁷ cells will need to be sorted. Another example is transfection experiments where other factors are involved (transfection efficiency, expression of dim target populations...). Based on the above, you have to make sure that you have enough cells to be able to sort and that you have a clean preparation to get an optimal sorting yield.

You also need to bring in:

• Culture media to add in the collection tubes
• PBS (filtered) in case we need to dilute your sample
• Micropipet and tips for homogenization of the sample before loading
• Your compensation controls (optional availability of beads compensation controls at the core)

Cell Dissociation and Filtration

​Cell clumping can result in poor sort purity when sorted target cells are attached to non-target cells and poor recovery when coincident aborts exclude all clumped cells. DNA from lysed cells in the medium can cause clump. DNAse will help reduce cellular aggregation. Cell clumping can also be reduced by filtering samples just prior to sorting by using various pore size disposable Filcon filter (ref #s: 340629 for 50µm and 340633 for 70µm).

Your Results

The generated data will be exported and burned on a CD/DVD. It will not be saved on a USB. At the end of each month, the operator will delete all the saved results to keep the database size minimal. You have to make sure that you back up all your data.

Quality Control: Baseline Report and CS&T

• The Baseline Report: measures key factors (linearity, resolution sensitivity, optimized PMT-V), determines fluorescence intensity for each parameter (PMT target value). It is done routinely every 6 months and after changes in the characteristics of the instrument (new lasers, new filters, adjustments during maintenance).
• Daily Performance Check (CS&T): measures variation from baseline from run to run, adjusts the PMT_V for each parameter to place actual PMT-value to target PMT-value). It is performed daily.

Note: If you are planning a series of experiments, it may be best to inquire about the baseline timing so that your results are comparable.

TIPS

Us​eful link for choosing fluorochromes/ tandem dyes

The following link​ can help you to choose the optimal fluorochrome combination for you experiment to reduce the spillovers and to get maximum signal possible.  Special care should be done when using tandem dyes (PE-Cy7, APC-Cy7, PerCP-Cy5.5, PE-Texas Red …) since these dyes are prone to degradation when exposed to light or to formaldehyde and their expression can vary from one lot to another.

Label-Specific Compensation

If your experiment requires compensation prior to sorting, then you need to provide proper controls for your experiment (single positive stained cells for each fluorochrome used) or you can ask for compensation beads from the facility manager (single positive stained beads for each fluorochrome used).

Creating an application for your experiments

It is advisable to create special application settings for your experiment if you plan to conduct the exact same conditions and fluorochromes over a series of experiments.

Sorting precision and gating strategy

For every sort, the sorting precision and the gating strategy should be clarified. BD FACSAria offers 4 different Sort Precisions: