Deborah, There a number of possibilities as to what's causing your bad luck. If you want some local help, I recommend talking to the Lefrancois lab there at U Conn - I know they've got the process down because I taught them how to do it. Here's the basic procedure we use. First, we do gradient separation to remove RBCs rather than lysis - lysing techiques tend to be pretty harsh even on PBMC, at least in my book. It's fine if you are just doing phenotypic analysis but for adoptive transfer you need healthy cells. Once you've got the cells, wash them in PBS or HBSS containing 0.5% BSA or 1% FBS. Count and resuspend at a concentration of 10 million cells per ml in the PBS/BSA or HBSS/BSA. Add 2 microliters of a 5 milliMolar solution of CFSE in DMSO (10 micromolar final). This part is crucial to cell survival - the DMSO needs to be of high quality and relatively fresh. I recommend making a stock solution of 5 mM CFSE in DMSO and aliquoting it out so you don't freeze/thaw the CFSE/DMSO more than once. Store the aliquots at 4oC, in a light-protected box. Once you add the CFSE/DMSO to the cells, let that go for 10 minutes at 37 in a water bath, then wash once with HBSS or PBS. Save a small sample of cells to check on the cytometer (although usually the pellets glow a nice yellowish-green) and immediately inject the cells into your mice. Transferring 1 million or fewer lymphocytes into C57Bl/6 mice we usually see nice CFSE-pos. cells for 2-3 weeks post-transfer, even in controls. Keep in mind that the cells do lose a substantial amount of CFSE in the first 24 hours post-labeling so they will not be anywhere near as bright as immediately post-label. I usually stick with the same settings we use for FITC, using a bright FITC-mAb for compensation. That usually puts the resting, undivided cells as a nice sharp peak close to the fourth decade on FL1. Good luck! Doug Douglas S. Reed, Ph.D. Microbiologist Respiratory & Mucosal Immunity Department of Aerobiology & Product Evaluation Division of Toxinology & Aerobiology U.S. Army Medical Research Institute of Infectious Diseases 1425 Porter Street, Fort Detrick Frederick, MD 21702-5011 301-619-6728 301-619-6911 fax doug.reed@det.amedd.army.mil -----Original Message----- From: deborah foss [mailto:dfoss@neuron.uchc.edu] Sent: Thursday, May 30, 2002 5:54 PM To: cyto-inbox Subject: CFDA-SE staining -- I just found this list while searching for reasons that the CFDA-SE staining is not working in our Laboratory. What we are trying to do is stain Peripheral Blood MC and inject them into the same strain of mouse. The CFDA-SE staining would be the way to tell the donor and host cells apart. We are using Balb-c mice. We sacrifice the donors, spin and wash the PBMCs, lyse the cells, wash again, count them, stain them with from 1uM to 10uM of the vibrant CFDA-SE made up as the package directs. We have tried incubating from 10 to 15 minutes, we have tried stopping the reaction as the directions said (wash, and 30 more minutes in the water bath). We have tried using 5%FCS to end the reaction. The cells are stained, we check on the Facs, they are all the way to the right on the log scale. Then we put them in the animal (IV), from 1 million to 5 million. When we take the tissues from the animals, (24 or 48 hours later), we don't see any staining on analysis with the Facs. We have checked both the spleen and the thymus. To check out if it was a problem with the staining, I irradiated the latest group of mice, 600r, 2 hours prior to the transfer. Still no green cells. I know that we are looking for a small population of cells, but I figure we have to be missing something. We are not looking for cell division, just adoptive transfer. Help! Debi
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