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Flow Cytometry Core Laboratory Equipment

  • Capable of "user-friendly" operation; investigators can be trained in instrument operation
  • One argon-ion laser tuned to 488 nm excitation
  • Capable of up to four fluorescent emissions
  • Typical emissions:
    • FITC
    • PE
    • PE-TxRed
    • PE-Cy5
    • PI
  • Analysis only instrument

  • Capable of "user-friendly" operation; investigators can be trained in instrument operation
  • Two lasers: an argon-ion laser tuned to 488 nm excitation and a HeNe laser tuned to 633 nm excitation
  • Capable of up to five fluorescent emissions
  • Typical emissions:
    • FITC
    • PE
    • PE-TxRed
    • PE-Cy5 or APC
    • PE-Cy7 or APC-Cy7
    • PI
  • Analysis only instrument

 

Cell Sorting
MoFlo high-performance cell sorter
  • One argon-ion multi-line laser,Coherent Innova 70-4
  • Capable of detecting four fluorescent emissions, as well as forward and side scatter
  • Capable of sorting at rates up to 50,000 cells/second
  • Capable of sorting 2 subpopulations simultaneously
  • Equipped with CyClone robotic arm which aids the deposition of sorted cells onto microscope slides or multi-well tissue culture plates
  • Run by a trained operator (Danielle King)

Cell Sorting Protocol
The MoFlo (Dako Colorado, Inc.) is configured with:

  1. one Innova 90-4 laser, tuned to 488 nm excitation
  2. forward and side scatter detectors plus 4 PMTs
  3. 2-way cell sorting at a rate of up to 50,000 cells per second (cps)
  4. CyClone, a robotic arm to directly deposit sorted cells onto microscope slides or dispense sorted cells into the wells of microplates

Scheduling cell-sorting experiments

  • Make an appointment with Danielle King at least one week in advance.
  • Please submit the sort form several days in advance so that the instrument can be configured for your sort.
  • If you need to cancel your sorting appointment, please do so as early as possible.
  • If you are running late for your sort appointment, please notify Danielle. Please keep in mind that charges for laser time begin when the laser is first fired up (usually just prior to your scheduled arrival time).

What to bring to the flow lab

  1. Your cells
  2. Extra media/buffer
  3. FBS/media (plan on 2-4 ml per 1 x 106 sorted cells) to add to sorting tubes
  4. A sample of unstained cells
  5. For multicolor analyses, compensation controls for each color used

Sorting speed. There are a number of factors that determine the length of the cell sort:

  1. cell type
  2. cell concentration
  3. sort mode [purity or enrichment]
  4. frequency of the target cells

For example, when sorting at a sample rate of 20,000 cells per second, 70 x 10 6 (take out millions) cells can be processed per hour. If the starting target cell frequency is 10%, those cells can be sorted out at a rate of 7 x 10 6 cells per hour. If the starting frequence is only 1%, only 700,000 per hour can be sorted out. Thus, it may be desirable to enrich the target cells of interest prior to cell sorting to increase the frequency of the target population.

Cells to be sorted. The flow lab is not equipped to sort cells with a BSL of 2 or higher. There is no containment system to protect the operator or any other lab personnel from exposure to the aerosols generated during sorting.

Count your cells immediately prior to sorting as this will allow us to estimate the efficiency of yield and to trace problems associated with a low yield. From experience, we have found that the staining process results in considerable cell loss, usually through centrifugation. We highly recommend that you prepare twice the number of cells needed for a sort. Immediately prior to sorting, cells will be filtered through nylon mesh (40-70 µm; available in the flow lab).

Cell media. Cells should be resuspended in a low protein buffer, such as Ca++/Mg++free PBS (or phenol red-free HBSS). The following media components are recommended to reduce cell aggregation when
working with white blood cells:

  1. Ca/Mg free buffer to prevent macrophages/monocytes from sticking to tubing
  2. 5 mM EDTA to prevent macrophages/monocytes from sticking to tubing;EDTA also helps to reduce cation-dependent cell to cell adhesion that sometimes occurs with suspensions of activated cells
  3. 0.5-2% BSA [not FBS]
  4. 1% heat-inactivated FBS [dialyzed against Ca/Mg++ free PBS]; <2% FBS
  5. DNase at 350-500 Kunits/mL; 10U/mL DNAase II to remove DNA-induced clumping
  6. 25 mM HEPES
  7. 0.01% hyaluronidase type V
  8. 0.1% collagenase type IV; Collagenase type II (Sigma)70 mg/25ml of serum free calcium and magnesium free HBSS/high glucose can be used to gently separate aggregated cells.

Number of sorted cells required. To help maximize cell recovery, we need to know:

  1. how many cells are present in your unsorted population
  2. the estimated frequency of the target population
  3. the number of cells that you require after the sort
  4. the desired result, high cell yield or high purity

Instrument sterility. Please notify Danielle if you require a sterile cell sort.
It is impossible to sterilize the instrument but we can take
precautions to minimize any potential contamination of the sorted populations. We highly recommend the use of antibiotics (pen-strep) if sorted cells are to be cultured following cell sorting.

Collection of sorted cells.
The sorted cells will be diluted in sheath fluid. We routinely use a sterile sheath buffer, a high-quality PBS solution that is
endotoxin-free and preservative-free from Dako. We can sort into
polypropylene tubes, microscope slides, or multiwell tissue culture plates. In all cases, it is best to sort into vessels with FBS/medium to help cushion cell deposition.

Post-sort Purity.
The purity of each sorted population will be tested after each sort. The purity of each sort can be compromised by several factors that are not due to instrument failure.

  • Staining problems: The cells of interest may not be completely resolved from the unwanted cells (overlapping populations)
  • Unwanted cells (or platelets/debris) may aggregate with cells of interest. These unwanted particles can disassociate after the sort, resulting in the contamination of the sorted cells
  • Loss of viability after the sort may result in cells exhibiting different light scatter properties from those of the original cells.
  • The capping of cell surface markers after the sort will often result in sorted cells that appear less fluorescent than the original selected population.

Recovery/Viability of sorted cells.
We have found that post-sort handling can easily lead to low cell recovery. In order to maximize post-sort cell recovery and viability, we recommend centrifugal forces of 1500 x g.

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