I have stored blood samples in EDTA for almost 6 months.Is't now suitable for the isolation of PBMC?
Dear Jamila,
Please consider these questions:
Did you freeze the cells or put them in the fridge?
What are the components of blood? Why do you add EDTA to blood samples?
If you froze them, what happens when you freeze/thaw cells without cryoprotectant and in an aqueous (watery) solution?
What are PBMCs? Are there cell types in PBMCs that proliferate and others that do not? Which cell types are you interested in?
If you had them in the fridge, what happens to cells if they aren't getting nutrients for 6 months?
That should get you your answer...
It is impossible after that much time. Cells will lyse and die if you store at 4 degrees. If you freeze again you will phase the RBC lysis and WBC lysis.
If you want to isolate PBMCs from blood use suitable histopaque solution and use fresh voided blood with suitable anticoagulant according to your nature of work.
Hello
agree with Balaji S N , this is impossible . i suggest follow this protocol and notes .
1. Blood collection – The following vacutainers (BD) can be used, 10ml volume
a. Green top – Heparin
b. Purple top – EDTA
c. Yellow top – ACD (acid citrate dextrose)
d. Note: Blood should be used within two hours of blood draw to ensure maximal cell
yield. Blood can be stored on the benchtop overnight for next day processing;
however, there will be some loss in cell yield. Never store the blood in the vacutainer
in the fridge.
2. Mix approx. 10ml of whole anti-coagulated blood with sterile buffer for a total volume of
20ml in a 50ml conical.
a. Buffer can be any balanced salt solution, e.g. PBS, Hanks' balanced salt solution
(HBSS), or media
b. Note: Buffer should be without Ca2+/Mg2+ to prevent cellular activation
3. Pipet 15ml of Ficoll-Paque PLUS (GE Healthcare) into a separate 50ml conical.
4. Gently layer the 20ml of diluted blood onto the Ficoll.
a. How good a PBMC layer you get will depend on how well the blood is layered on the
Ficoll. There should be minimal if any mixing of the two liquids.
b. Using a 10ml serological pipet and pipet controller (e.g. Pipet-Aid) works best. Keep
the pipet tip on the side wall of conical and let blood slide down onto the Ficoll.
c. Use the least amount of pressure on the dispense button to layer the blood, better
yet if the dispense button has different speed settings – set on the slowest speed
possible
5. Spin sample at 400xg for 30-40min at room temperature with NO BRAKE.
6. Collect PBMC layer at diluted plasma/Ficoll interface.
a. Try to collect as little Ficoll and diluted plasma as possible.
7. Add buffer to PBMC for a total volume of 40ml.
8. Spin at 200xg for 10min at room temperature.
a. This is important to remove any contaminating Ficoll and platelets/plasma proteins.
9. Repeat Step 7-8.
10. Count cells with hemocytometer.
Note: This protocol was modified from the instruction sheet for Ficoll-Paque PLUS. All solutions/buffers used should be at room temperature prior to starting procedure.
Note: Monocyte purification from the PBMC can be done using magnet assisted cell sorting(MACS). Miltenyi is the company that has been used in the lab. All cell separations havebeen done following the provided instructions that come with the sorting beads.
Good Luck
It depends, how you stored them. I would not put efforts in Isolation, if they were stored at -20 C in EDTA. Preservation requires deep-freezing (-70) with c.f. glycerol.
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