I am trying to isolate mono nuclear cells (MNCs) from cord blood (which used to be my routine job during PhD) but I am not able to get the MNC fractions (buffycoat) now. One of my observations is that the ficoll-paque I am using expired three years ago. I have never come across this situation before. So please clarify my doubt. Thanks in advance
Salam alikum
Doubts is true , use Ficoll-Paque should be within 12 weeks of opening , or before the expiration date as per the manufacturer’s instructions.
MNCs are isolated bydensity gradient centrifugation using Histopaques-1077 (Sigma,St. Louis, MO) . Briefly, cord blood is layered over Histopaque (1:2 v/v) and centrifuged a t400g, for 30 min, to separate the MNC layer at the interface. MNCs are then incubated in RBC lysis solution (Sigma, St. Louis, MO) and then washed once in DMEM:IMDM medium. MNCs are then seeded at a density of 3 103 cells/cm2 in DMEM containing 10% UCBS in a humidified chamber at 37 1C and 5% CO2. Cells were then passaged every 7 days with a split ratio of 1:2. We optimized growth medium for MNCs using MTT assay. MNCs after isolation were seeded at 2000 cells/well in 100 ml of respective medium in quadruplet in tissue culture-treated flatbottom 96-multiwell plates. Each plate is then taken at predetermined time points (days 1, 2, 3 and 7) and 10 ml of MTT (5 mg/ml in PBS pH 7.4, filter sterilized) was added to each well . Plates were incubated at 37 1C in 5% CO2 for 3 h. Formazan crystals formed were dissolved in acidified iso-propanol and spectrophotometric reading was taken on spectra max 250
Good Luck
In my experience, I ued it for more time and I dnt have troubles.
Your doubt is true, with three years of elapsed time it will definately affect your separation better get fresh and not expired Histopaque to use as you are wasting your time and other resources.
Hi Dr. Otaso,
You mean "more time" over 3 years? We have some Ficoll which have been expired four years ago. Is it ok if we use them or not?
Hello Arihant, If you don't want to discard expired ficoll, use higher ratio then your blood sample. Since the principle is density gradient separation, more ficoll can give you better separation. If you have red blood cell contamination, change the media more frequently until you get rid of RBC. Hope this help
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