Hello, Am a M.V.Sc student from Madras Veterinay College currently...

I'll follow your instructions. Thank you sir.

I'm not sure I completely understand the step at which you are seeing clumps. Is there a reason you are not pelleting? There will always be some debris and clumping in the prep, but after you pellet, you can resuspend the clumped cells into media (see Part 1. A. Step 14) into as much or as little media as you need to make a dilution that gets you an ideal number of cells per well after mixing with matrigel.

Sir, Thank you so much. Actually after filtering only it is getting clumped before mixing it with matrigel. am adding the cells to the matrigel without pelleting it, so the number of cells is very less and it varies with each well. what may be the reason for the cells to get clumped?

Hi Kaviyarasia,

I do not have experience working in chicken crypts. I would recommend the following steps to prevent mucous accumulation:
1- during the initial wash (Part I, A.3) perform the PBS wash thoroughly. There should be no visible debris in your tube after washing. This is usually 5 washes per mouse colon.
2- Be sure to add DNAse to your suspension after EDTA/Collagenase treatment. This will help prevent DNA from clogging the filter and increase your yield.

Let me know if this helps! Best Regards, Kevin.