Hours of operation
: 8am- 5pm
Dr. Samia Khoury
Ms. Maria Esmerian
: DTS, 3rd floor, room 308-C
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.
Specifications of FACSAria SORP |
|FSC||488|| || || || ||Photodiode|
|SSC||488|| || || ||488/10|| |
|Alexa Fluor 488||488|||||||||||
|PI (Propidium Iodide)||488||536||617|||||||
|Pacific Blue||405||401 ||452||-||450/50||405-F|
|BD Horizon BV510||405||402||510|||||||
|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|
|Hoechst Red||355|| || ||600LP||670/30||355-A|
FACSAria SORP Cell Sorter Software
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
- 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)
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).
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.
Useful 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.
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:
|Purity||Best purity possible||Efficiency is affected (often <80%)|
|Yield||Efficiency is always 100%. Sort rare events||Purity is affected (often <90-95%)|
|4 Way Purity||Same purity as “Purity" precision, but the far side streams are better focused.||Efficiency is sometimes slightly lower than using “Purity"|
|Single Cell||Sort into plates (not only single cell cloning)||Massive loss of cells of interest|