I'm trying to set up a protocol for PBMC isolation from whole blood using density gradient centrifugation. In a previous lab we diluted the blood in RPMI prior to layering, and washed the PBMCs in RPMI, but a lot of protocols I've seen use PBS instead. Is there any reason to use one or the other, or do they both perform the same function?
You have not mentioned what you do with the PBMCs after the isoloation. I have used PBS for all my PBMC isolation and has worked well for the purposes that I have required them that is flow cytometry and immunocytochemistry. Addition of 0.5% Bovine Serum Albumin (BSA) helped increase the viability in flowcytometric studies. I personally have no experience with using RPMI but if you need to culture those cells post separation then maybe using a tissue culture medium may make a difference in the final outcome.
I generally use a BSS solution to dilute whole blood (1:1).
Solution A
Anhydrous D-glucose 0.1% [1.0 g/L]
CaCl2 × 2H2O 5.0 × 10^-5 M [0.0074 g/L]
MgCl2 × 6H2O 9.8 × 10^-4 M [0.1992 g/L]
KCl 5.4 × 10^-3 M [0.4026 g/L]
TRIS 0.145 M [17.565 g/L]
Final Volume 1 L, pH is 7.6 (with 10 N HCl)
Solution B
NaCl 0.14 M [8.19 g/L]
Mix 1 volume of A with 9 volumes of B.
Viability will be high and You can use cells for any downstream application (i.e. lymphomoncyte cultures).
You can use also PBS, but it must not contain neither calcium nor magnesium
I have no experience with RPMI in this step. Usually, I used PBS for PBMC isolation by gradient. I separate the PBMC in the short time after that will suspend in the medium, PBS, PBS-BSA or RPMI depend on the test.
Depending on what you are going to with cells afterwards, both solutions can be used. You need to be careful with PBS if you are going to do immunophenotyping of cells. If not prepared well and stays a while in lab, and if pH changes from 7.2 - 7.4 there can be crystallisation which effect your results. Our practice is to use RPMI if we are going to culture cells.
Crystallisation and bacterial contamination in case BSA is added are problems with PBS that has been stored and can wreck your results. We have PBS as two separate stock solutions and make fresh working solution for each day.
Thanks for the answers everyone. It sounds like there's no reason not to use RPMI, and since I've used it before and I may want to culture the cells I think I'll stick with RPMI.
It doesn't make any difference whether u r using PBS or media RPMI
NycoPrep works fine, since it is completely inert and does not disturb the cells.
The last wash is preferred in RPMI for many tests on Lumniex platform. If you are sure about the quality of PBS then dilution and initial washes may be in PBS.
PBS is adequate, if you are going to layer them and do the usual gradient centrifuge separation, however cells will be more nourished with Glutamine from RPMI, if you are using medium. Nonetheless, if you are going to separate T & B cells, you might want to use any physiological buffer or incomplete RPMI medium (not supplemented with FBS+antibiotics) as this might give a low yield of B cells.
You need PBS without Ca and Mg if your blood is drawn on EDTA collection tubes for gradient density separation and for the two first washes .If your blood collection tube contain heparin you can use PBS or RPMI.
We are also using PBS for diluting blood before layering and getting a good buffy coat layer. We never used RPMI for dilution.
please what is the essence of using the RPMI-1640 to wash the PBMCs?
Using RPMI is recommended if the cells are to be cultured and check for viability since RPMI will enhance PBMC yield
Usually RPMI provides an appropriate culturing conditions, it helps control bacterial contamination
BD Biosciences also recommend the use of IMDM (Iscove's Modified Dulbecco's Medium) for diluting whole blood samples prior the antibody staining for flow cytometry analysis.
Reference: the manual of "BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (Cat. No. 554714)" page 17. www.bdbiosciences.com
- Badges
- Science method