After last centrifugation of washing, resuspend your pellet in 1 mL culture media/PBS. Now take 10uL of this cell suspension and mix with 90uL of PBS.

Further, take 10uL of this diluted cell suspension and add 10uL of trypan blue. Mix well by pipetting.

Now charge your haemocytometer with this solution and count your cells in all the 16*4 squares. Sum the counts and take average.

Calculate the Concentration=

Average*dilution factor (20 in this case)*10^4 cells/mL.

• Yes. Like Jitendra Kumar Shandilya says, resuspend the pellet well in 1 ml of the culture media by gently pipetting up and down. Before the cells settle down, transfer 10ul to a fresh tube and add 10 ul of the trypan blue.
• If the pellet is small (less cells) take 10ul in 10ul trypan blue or 50 ul of cell suspension and 10 ul of trpan blue. If the pellet is big (more cells) then you can take 10 ul of cell suspension in say 190 ul of trypan blue.
• Mix throughly and gently and use 5 ul for the counting
• The purpose of having such variations is to have a final range of 30-70 cells in each quadrant of the hemocytometer while counting. too less cells or too many cells increase the chance of error in cell counting.
• Depending on the volume of cell suspension and volume of trypan blue, the number of cells can be calculated as
• Average number of cells in four quadrants * dilution factor which is (volume of cell suspension+volume of trypan blue)/volume of trypan blue
• The result will ....... x 10^4 cells
• Make sure to give a proper time gap between addition of trypan blue and counting (as per literature) to eliminate the dead cells while counting.
• Standardize the volumes of cell suspension and trypan blue and waiting time for each type of cell and follow the same in all experiments

Best,

Hello, Ayah!

As mentioned above, you should add PBS up to 1mL at first. I add just a little over 1mL just to make sure i have 1mL left after i take my sample for cell counting.

However, if you can and have the means in your lab, i strongly recommend you use an automatic cell counter. Using trypan blue and manual counting takes time, effort and is not the most accurate method, being especially prone to errors due to interindividual variability. A machine is only subject to it's internal variability (imprecison of measurment).

I think that any automatic cell counter could work, you don't need an expensive one. We have a lot of experience with isolation of PBMC from heparinized peripheral blood and we do the initial counting from the whole blood and also the counting from the resuspended pellet using the same automated cell counter. This also helps monitor your isolation performance as you can easily calculate the yield.

I'm glad to have answers from you all!

thank you so much Jitendra Kumar Shandilya but in your example the D.F is 2 not 20 right?

Aditi Devi N many thanks for you but D.F is total volume/ sample volume (cell suspension not the trypan)

Ion Bogdan Mănescu thanks a lot, can i use a CBC machine since we don't have cells counter?

Ayah Alkhaldi Its 20 as your cell suspension is diluted 10 time first in PBS and 2 times in tryphan blue, so its (10*2= 20 time dilution).

@Ayah Alkhaldi

Yes, a CBC machine is great. I was talking about "cell counter", but i was refferring to a CBC machine. We use a simple SYSMEX XS-800i which is more than enough for our needs.

Also, you may want to investigate whether you can do a CBC from a pellet resuspended in colored culture medium (RPMI for example). I have never tried it, but have a vague feeling that it's not ok because of the red color. Counting from PBS suspensions is ok.

Moreover, i remember that when we first tried to do a cbc from PBS suspension, the machine returned an error as it expected the aspirated suspension to be red (it was meant for blood). So we had do deactivate that from the menu. Hope it won't be the case for you.

Thanks alot for you all. I appreciate your efforts!

Hello Everyone,

If you do not mind, I want to chime in with a question. How do you determine which cells to count after PBMC isolation? We use trypan blue with a 1:10 dilution factor to count our cells with a hemocytometer. I notice under the microscope there are various types of cells (larger and smaller). Do you count every cell in your counting? We are trouble shooting with OptiPrep and Histopaque and get clean bands with both. The lab I work in doesn't have much experience isolating PBMCs and I think we are hitting a road block with the cell counting for our flow cytometry experiments. Thank you for your time.

Cesar De Jeronimo Diaz

Don't worry about PBMC isolation, you just need to go on. Only by experience can you get good yield and purity. We've been using Histopaque and Ficollpaque, it doesn't really matter the brand. We have lots of experience with isolating PBMC, but i have to admit it wasn't easy in the beginning. However, we pressed on and now we have very good performance i.e. 65-90% yield, 95-99% purity routinely. I will add some piece of advice although you may already be aware of these aspects:

1) Make sure your Ficoll solution is at room temperature (we usually remove it from the fridge 30 minutes before isolation. Density is temperature-dependent.

2) Do dilute your blood with PBS at least 1:1.

3) Make sure you gently layer the diluted blood on the Ficoll layer. Keep the tube tilted and reposition it vertically a little at a time. The first mL is the most difficult. To deccelerate the diluted blood column coming out of the pipette, eject the diluted blood on the inner wall of the tube and it will just slowly drain on the walls.

4) VERY IMPORTANT: make sure the centrifuge has the brakes off. The PBMC band can get disturbed and mixed with the other layers if the centrifuge has the brakes on.

5) Carefully handle the tubes after centrifugation.

You may want to check out our article about the isolation of PBMC. We played around with some of the isolation parameters and we are very pleased with our current protocol. Most importantly: keep exercising, practice makes perfect!

Article Optimization of a Density Gradient Centrifugation Protocol f...

Cesar De Jeronimo Diaz I count large ones.

Ion Bogdan Mănescu I didn't use the separation media that you mentioned, instead we use lymphosep media. should i store it in the refrigerator although the manufacture recommend to store it at 25C?

Also, should I care for cell viability after trypan blue staining even if I will not culture them (we only extract protein)?

@Ayah Alkhaldi

As long as you get the job done with Lymphosep, everything is fine. I don't think you should store it in the fridge if the manufacturer says room temperature. In our case (Histopaque-1077 from Sigma Aldrich), the manufacturer says to store it at 2-8 °C. That is why we keep it in the fridge, but need to get it to room temperature before usage.

As for cell viability... I see no apparent reason to care for viability if you are only interested in protein extraction. Just don't take too long between viability testing and protein extraction! Trypan blue is known to overestimate cell viability. You would normally not consider early-apoptotic cells as viable, but trypan blue does cannot differentiate between apoptosis and necrosis.

That is why you should work fast. Who knows how apoptosis/necrosis may influence your proteome and therefore your data. Generally, it's better to have as few variables as possible so when something goes wrong, you only have a couple of suspects to look into. Anyway, i don't think this is a real problem, but you know... It's good practice :)

Ion Bogdan Mănescu thanks a million for your helpful notes. i still have one question, when i centrifuge my samples the plasma color is bright pink so does it because we are using RPMI instead of PBS to dilute the blood?

Ayah Alkhaldi

You are welcome. Glad whenever i can help.

Honestly, i've never diluted the blood with RPMI before isolation, but most probably, the plasma is pink because of it.

Funny story about colored plasma (which i hope you will never have to come back to for troubleshooting):

After hundreds of isolations using the same reagents, a new lot of PBS was sent to us by the same provider. We had three rounds of failed PBMC isolation, the plasma was reddish, the PBMC band was barely visible and a CBC and microscopy were telling us we had general cell lysis. I was scratching my head and not finding the problem. However, we did some testing and everything pointed to the PBS as being the only responsible. When i watched the label, it said PBS 10x. You can imagine the lols in the laboratory after that :)))

We were so used to being supplied with 1x PBS, but this time the manufacturer sent us a 10x flask and we didn't bother to check the label. It was funny but also a valuable lesson...So yeah, those were the only times my plasma was colored reddish/pink, haha, and i don't wanna see it again.

Ion Bogdan Mănescu

thanks for sharing your previous story. I hope you will not face any problem like that!

Ayah Alkhaldi Yes ! you are right

its total volume / volume of cell suspension

apologies !

Aditi Devi N never mind.

many thanks!

Thank you all for this it is very helpfull

after isolation of PBMCs you can use trypan blue method but this is time consuming, and you have only less than 15 min to do your count before cell lyse.

I recommend using automated CBC device to count the cells.

I did it and it works >> you just select the higher precision with 5 differential apparatus

All, I work for a small Clinical Lab in a CRO and we are adding an automated cell counter for PBMC analysis for a specific Clinical Trial. I have no experience with a hemocytometer beyond schooling 20+ years ago.

Would any of you be willing to be a reference for me to ask questions? I really need some guidance on this subject.