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CFSE-labeling for in vivo Monitoring of Adoptively Transferred Cells

By Stanley Morris,2014-02-08 06:35
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CFSE-labeling for in vivo Monitoring of Adoptively Transferred Cellsof,in,cells,vivo,for,Cells

CFSE-labeling for in vivo Monitoring of Adoptively Transferred Cells

    1. Prepare lymphocyte suspension in 5 ml RPMI with 10% FCS

    2. Count cells

    3. Prepare 5 ;M CFSE

    a. Add 10 ;l of 10 mM stock CFSE into 20 ml HBSS (serum free)

    b. Equilibrate at RT in the dark for 10 min

    4. Spin cell suspension 5 min at 1400 rpm; remove media 65. Resuspend cells at 5x10/ml in 5 ;M CFSE (when resuspending break up pellet with 200

    ul HBSS [without CFSE] then add total amount of CFSE directly). The addition process

    is very important if you wish to track dividing cells because you need a very uniformly

    stained population.

    6. Incubate at RT in the dark for 8min (exactly; do not go over with this step)

    7. Immediately add 20 ml (or equivalent staining volume) of HBSS/20% FCS and spin

    cells for 5min at 1400 rpm 4?C

    8. Remove media and add 20 ml of HBSS (serum free); spin cells for 5 min at 1400 rpm

    9. Repeat wash

    10. Count cells with trypan blue to verify viablility 711. Resuspend CFSE labeled cells at 2.5x10/ml (or at appropriate concentration for your

    adoptive transfer experiments) in HBSS (serum free)

    12. Pass cells through 40 ;m filter to remove any cell aggregates

    13. Keep cells in foil on ice until adoptive transfer of 200 ;l IV with 28G? insulin syringe

    a. Adoptive Transfer: perform remainder in animal facility procedure room

    b. Heat beaker of water on hot plate to warm (you should be able to touch without

    pain for a short period)

    c. Put mouse into illuminated mouse holder for IV injection

    d. Soak gauze pads in water and then wrap the tail for 10-20s with gauze

    e. Remove the gauze and proceed immediately with injection

    *Be sure to remove some cells prior to and after labeling to verify CFSE incorporation by flow.

    *The presence of FCS in the staining buffer inhibits incorporation of CFSE but adding HBSS with serum immediately after is needed to maintain the viability of the cells after staining as CFSE is highly toxic to cells. After serum rescue it is essential to remove all serum prior to transfer.

    *The concentration of CFSE may need to be adjusted depending on its application. You may wish to drop the staining concentration to 2.5 ;M if you are having difficulty with toxicity (2.5 µM is probably the first one you should try). If you wish to track cells in vivo for extended times you may need to increase the CFSE. However, this may also lead to increased toxicity and subsequent 7adjustments such as decreased incubation time or increased cell concentration (10/ml). The

     10 ;M. recommended range is 1 ;M

2 Kits available

     Vybrant CFDA SE Cell Tracer Kit (Molecular Probes, Cat # V-12883)

    o 10 vials lyophilized CFSE at 500ug/vial

     CellTrace CFSE Cell Proliferation Kit (Molecular Probes, Cat # C34554)

    o 10 vials lyophilized CFSE at 50ug/vial

I recommend the smaller kit as CFSE is not very stable and toxicity rapidly increases during

storage. Do not use CFSE after 6 months at -20?C.

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