What is a composition of your medium and size of channels you put cells through?

Try adding some dextran 40 to your media at about 20-40mg/L. Heparin can cause RBC aggregation so don't use it in the media that you use in the microfluidic device. You can wash your RBCs in PBS (300mOsmol/kg pH 7.4) twice and resuspend in PBS with dextran 40. You could also try increasing the shear in the device as well (higher flow rate or smaller dimension in part of the fluid path) to disaggregate the cells. RBCs often aggregate in low shear environments.

You can prevent RBC aggregation in  BUFFER by two ways:

1. FULL removing of proteins from suspension medium by repeated washing;

2. Increasing of flow shear stress (or shear rate).

If you're planning to carry out measurements in PLASMA, you should follow the Ron`s recommendations (manipulation with dextran 40). 

Medium is Sucrose (%8.5w/v) and Dextrose(%0.3w/v) mixture and channel dimensions are 1000um width and 40um height.

Thanks for your kind answers. I will try dextran 40 as you explained above.

Gurhan,

In order to prevent aggregation of RBCs in microchannels, I diluted RBCs in PBS containing EDTA (Ethylenediaminetetraacetic Acid) for anticoagulation. Then RBCs were washed three times by centrifugation and re-suspension of the pellet in GASP buffer (PBS + Bovine Serum Albumin). You can find the details of the protocol on http://www.sciencedirect.com/science/article/pii/S0026286212001550 section “RBC suspensions”.

Regards

Sophie,

Did the RBC's crenate when GASP buffer was used. We normally use 1x PBS which would keep RBC's intact for imaging. But when we prepared GASP most of the RBC's crenated. Any suggestions? Thanks in advance!

Hi Jayesh,

No, the RBCs were not crenated in GASP buffer. This can be an artifact of the EDTA (we do not use EDTA in GASP solution).

Did you checked the pH of your GASP? It must be around 7.4 (maybe the pH of the water you are using is too low or too high). If needed, you can adjust the pH with HCl for example.

Hello Sophie,

We are trying to flow Whole blood in microfluidic channel. We have to dilute the whole blood with GASP buffer say in the ratio 20uL Blood : 970uL buffer : 10 uL MB stain. With respect to the above mentioned link, how different should the Glucose concentration be? We observed that cells were crenating and we suspect it to be glucose concentration issue. Thanks in advance 

Hello Jayesh,

The glucose concentration in the GASP buffer is supposed to be similar than the concentration in plasma and should not be a problem here. You can try to make a buffer with no glucose to test it. Again, have you checked the pH of your buffer? This may be the issue.

In addition, why are you using MB stain? Do you think that can affect you solution?

Cheers,

Sophie

You can collect blood in CPD ( Citrate phosphate dextrose ) and wash them properly with ice cold PBS . It will work well.