PBMCs mostly consist of lymphocytes about 60-70%. In these 5 of CD3+ T cells is nearly 70-80%.
In my flow cytometry experiments, the most yield that I can get is 30-40%. For optimization, I have changed the Ficols (GE, Hisep), have changed the PBS for diluting whole blood, even tried using RPMI instead of PBS, handling issue was also checked and donor of blood sample was also changed.
The protocol that I was using was 1:1 dilution of whole blood with PBS, Layer on the Ficol: diluted blood in 3:4 ratio. Centrifuge at 400g (also tried 550g) for 30min at max acceleration and no break. Harvest PBMC layer. Wash with 3x PBS at 2000rpm for 10min twice.
After all the optimizations, the T cell yield is still 40%.
Cell viability assessed by 7-AAD is 90%.
Kindly help me with the any further changes that can be made to increase the yield.
My suggestioons is to use :
1. Lymphoprep™;Density gradient medium for the isolation of mononuclear cells instead of Ficol. ficol is toxic for cells
2. add 15 ml Lymphoprep and hold the tube with 45 grade and then add diluted blood as quick as you can but carefully with mixing.
3. use the good pipette-boy which you can control pipetting well and quick.
4. use higher centrifuge speed and I use to have 760g but little less acceleration 7. my centrifuge MAX acceleration is 9.
I hope these tips help you to get higher yield.
Best regards
//Bahar
Might be a silly answer but: Why not use clinically prepared hospital tubes? The amount of washes is reduced (maybe losing lymphocytes each wash?) and the 'buffy coat' is better. I've never been a fan of FICOL as it needs really careful preparation/loading/settling and layering and we tend to go too fast when seting it up. My old faourite is to tell students 'set-up' the gradient and go for a coffee and then layer the blood' We sometimes go too fast when we prep some samples.
Thanks for answering Maria Cinta Cid Xutgla Bahareh Khalaj
Although I havent used lymphoprep, I used Hisep apart from Ficol But i got the similar yield.
I'll try with lymphoprep too.
What is the aprx % yield of Lymphocytes do you get?
Brian Telfer Thanks for replying!
As i work at a non-clinical setup, getting fresh blood is rather easier than buffy coat.
Also, I did not understand the meaning of "set up", Please clarify on that.
Thanks
Hi Varsha,
I get aprx 70% lymphocyte. one thing may be it is silly but I want just mention it. which Temprature they keep the Whole blood before you get and when they prepared? if the whole blood is keeping for more than 4 hour in the +4 you don't get enough cells. you loose every thing. I agree to the Brian it is better to get Buffy coat from blood bank which is prepared in the same day and it almost fresh. In the buffycoat you should do same procedure as whole blood. I hope it helps.
ok. Whole blood is processes within 2h of collection. and before isolation blood and hisep/ficol both brought to room temperature.
the cell viability is same by ficol and hisep and is more than 90%.
Im getting only 30% lymphocytes.
Thanks for your inputs Bahareh Khalaj
Hi Varsha, 'set-up' means to have everything in place at the same temperature and allowthe ficol prep to settle before going striaght into the next step of the separation process. By allowing the sample and ficol to 'rest' for a little while it allows 'all' the components to reach an equilibrium of temperature before spinning down in a centrifuge.
In other words don't go through the steps too quickly until 'equivalence' of temperature is reached and layer the blood gently down the side of the tube at a 30 degree angle if using Ficol (this might reduce the disturbance of the top layer?). A possible problem I have seen in the past is when the ficol gradient has not settled and a 'vortex' is created when the sample of blood is layered very fast onto the surface of the ficol and the sample is loaded directly onto the top of the ficol gradient rather than gently loaded by keeping the blood sample about 1cm from the top of the ficol gradient at a 30 degree angle and then gently put into an upright postion when gentle application of the sample has been carried out. Hope this helps.
May be silly, but have you made sure the 'brake mechanism' on thecentrifuge is 'switched OFF' during the centrifuge process?
Dear Varsha,
I'm not sure whether this is of interest to you, but if you want to isolate specifically CD3 T cells directly from whole blood or buffy coat with a high yield, I can recommend the CD3 Fab-TACS® Isolation Kit. You can directly apply e.g. whole blood, but also PBMCs to the columns and positively select CD3+ T cells. In a final step, the labeling is removed, so you get label-free cells. You can find more information here: https://www.iba-lifesciences.com/fab-tacs.html
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts