Hello!
It's me and Embryoid Bodies again.
When it comes to total protein or mRNA isolation from EBs it's fine. But I have to admit that BCA shows low amount of total protein after lysis... But it still works.
When I want to prepare EBs for flow cytometry analysis and obtain single-cell suspension without losing cell viability, it gets really difficult.
I have never ever used EBs this way before so I am using protocol typical for adherent/suspension cells and it goes like this:
1. Centrifugation at 4 500 RPM for 4 min at 18*C.
2. Washing cells with PBS twice. Each time centrifugation at 4 500 RPM for 4 min at 18*C.
3. Adding 100uL accutase + DNase, incubation for 5 min.
4. Washing cells with PBS to eliminate accutase.
5. Adding 100uL Binding Buffer and resuspending the pellet.
6. Adding antibodies and PI.
7. Incubation for 10 min in RT in the dark.
8. Centrifugation at 4 500 RPM for 4 min at 18*C.
9. Resuspension of the pellet in 100uL Binding buffer.
This protocol is used in our lab to analyse typical adherent/suspension cell lines.
I am not sure if this protocol is also good for EBs. The pellet obtained after centrifugation is almost impossible to be resuspended... It feels like such pipetting kills cells and there's nothing to analyse.
Do you have any tips? Or maybe you had already done such analysis using EBs?
Thank you for your help
hello ..
Embryoid bodies (EBs) are a bit different from typical adherent or suspension cells, as they are formed by aggregated cells that have undergone differentiation. The protocol you've described is generally used for dissociating cells from a monolayer or a suspension, but it may not be the best approach for EBs, as they are structurally more complex.
There are several different methods that have been used to dissociate EBs, each with its own set of advantages and disadvantages.
One common method is to use a combination of mechanical and enzymatic dissociation. This can involve gently triturating the EBs with a pipette and then adding an enzyme such as trypsin or collagenase to complete the dissociation. This approach can be very effective in breaking down the extracellular matrix and releasing the individual cells, but it can also be quite time-consuming and labor-intensive. Additionally, it is important to note that using enzymes will cause cells to lose their viability if the EB are not rinsed and washed correctly after.
Another approach is to use a mechanical dissociation method, such as using a cell scraper or a cell dissociation buffer. These methods can be faster and less labor-intensive than enzymatic methods, but they can also be more harsh and may result in a higher rate of cell death.
In any case, it is important to optimize the dissociation conditions for your specific application and use the most mild and efficient method possible. The optimal conditions are going to be depending on the time, volume, temperature and the equipment you have access to. I would recommend testing different methods and varying the conditions to see which one works best for you.
It may be also helpful to try different types of buffer and centrifugation settings, as well as varying the timing of the step to see what works best for your sample. Also, you can try to manipulate the EB prior to dissociation, like freezing and thawing, trypsinization or chemical treatments as well.
In any case, it is important to be careful and gentle when handling the EBs and to keep an eye on cell viability throughout the process to make sure you're not losing too many cells. Flow cytometry analysis of EBs is relatively difficult and might require to optimize the technique as best as you can.
Regards.
The only thing I would change here is substitute Accutase for AccuMax. It's a higher concentrated version, more effective for getting single cells. If that's not enough, shear through a narrow syringe needle. And finally filter through a 30 um cellstrainer.
- Badges
- Science method