Stanford Medical School Blood Center has buffy coat human blood to sell but I would like to get it locally. We tried to ask around and never got the answers. It doesn't need an IRB approval since no subject info is required. We contacted Tampa blood bank and they don't know either.
I have purchased human buffy coats from Innovative Research Inc. Its in Michigan, but the cells were OK after ON shipping.
http://www.innov-research.com/product/buffy-coats-leukocytes/
If thats not ideal, you can always make the buffy coat yourself from whole blood.
If you're willing to make it yourself (only need a centrifuge really), a blood bank should be willing to give you a sample of expired blood that they are going to throw away. I've done this with a hospital on my University campus but there were no complications, IRB etc.
Thanks, Steingrimur & Simon, for your info.
We want to get mononuclear cells (for cell culture) from either buffy coat or whole blood but just cannot find a way to get them.
If you want to isolate specific cells from blood you can use kits from Miltenyi.
https://www.miltenyibiotec.com/
Its very easy to isolate buffy coat from blood, what you need is ficoll and there are few protocols known for isolation of buffy coat with the help of ficoll.
I used to collect PBMC from blood, so I used Ficoll for isolation of buffy coat from blood.
What ever steps I followed were in ice.
_Take blood (suppose 4 ml ) then add equal amount of PBS or RPMI ( 4 ml).
_Take Ficoll in two falcons, 4 ml each. Ficoll is very viscous so its difficult to balance it during centrifugation, thats why we need to take 4-4 ml in two falcon.
_Add diluted blood (same volume of ficoll) 4 ml, in falcon containing Ficoll, but add very slowly...(this diluted blood should form a layer on ficoll, blood should not disturb ficoll) its always preferable to add diluted blood very slowly from aside.
_Then take these two falcon to the centrifuge in ice and centrifuge it on 2000 rcm for 45 minutes at 0 - 4 C.
_ Then you can easily take out the buffy coat in falcon and wash it thrice with PBS. 5 min each on 8000 RPM ( 0C - 4 C).
_ Take the pallet and re suspend in RPMI 10-15 %.
If you're trying to collect common cells of buffy coat , you can take peripheral blood about 4ml in a 12 mm x 75 mm falcon tube with an anticoagolant (e.g. Heparine). Then centrifuge it on 1600RPM / 10min. using a pasteur pipette you can collect this ticky buffy coat very slowly. although i'd never used Priyanka Srivastava protocol but i think it's a very good idea.
Thanks, Priyanka and Najmeh, for your detailed protocol, and Nabi for your suggestions. We'll try your protocol
A centriguge and a pipette is all you need; no requirement for a fancy procedure. Im sure your lab can show u how to do it in 5 minutes. Sometimes the simplest solutions are the least obvious. Hope that helps.
Thanks Robert for your suggestion. Do you know where and how to get human whole blood first or buffy coat blood?
Buffy coat can easily be obtained from peripheral blood which should be placed in a citrated tube (one part of sodium citrate to 9 parts of whole blood). Mix well. Centrifuge the citrated blood at 3000 revolutions per minute for 20 minutes. After centrifuge, notice that at the junction of the plasma and red cells, there is a thin whitish or buffy layer. This is called buffy coat. First separate the supernatant platelet poor plasma and then using a pipette, separate the buffy coat.
NB: @Omer: "Centrifuge the citrated blood at 3000 revolutions per minute" is data of no value except you give information on the centrifuge you were ussing. Usually (for use on ALL types of centrifuges) one reports the g-forces achieved (i.e., e.g. 1000 g or 2.500 g, etc.) independent of rotation/min(rpm) or type of centrifuge rotor (and its diameter)....
Also please: cf. the old thread
https://www.researchgate.net/post/Does_anyone_have_experience_with_buffy_coat_collection?
Thank you
Hi Dr. Wang,
Did you get blood from Tampa blood bank? which one? Please let me know. Thanks in advance.
Best regards,
Jinesh.
Buffy coat preparation protocol
- Add an equal volume of recommended medium to whole blood. ...
- Centrifuge at 800 x g for 10 minutes at room temperature (15 - 25°C) with the brake off.
- Remove the concentrated leukocyte band (this is the buffy coat), plus a small portion of the plasma and concentrated red blood cells (RBCs).
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