I have thawed my cells 3T3 (fibroblasts) a couple days ago. After thawing I added 9ml of warm media into the cells and centrifuged them at 1000rpm for 5minutes. I sub-cultured my cells twice but why I am seeing black pellet of cells after centrifuging during the subculturing? But my cell viability is 90% when counted on second sub-culturing using hemacytometer.
Aneela Davuluri Observing a black pellet of cells after centrifugation can be unusual and concerning, especially when working with 3T3 fibroblasts. However, the fact that your cell viability is still high (90%) is a positive sign. Here are some potential reasons and considerations for the black pellet observation:
Steps to Address the Issue:
- Microscopic Examination: Examine the pellet under a microscope to check for signs of contamination or unusual cell morphology.
- Assess Reagents and Media: Ensure all media and reagents are fresh and not contaminated. Check if any supplements or additives could cause discoloration.
- Repeat with Fresh Reagents: If possible, thaw a new vial of cells and repeat the process with fresh media and reagents to see if the issue persists.
- Contamination Test: Conduct a sterility test on your media and cell culture to rule out microbial contamination.
- Environmental Factors: Consider environmental factors such as incubator conditions, temperature fluctuations, or exposure to light.
Hope I have addressed your query, if you have any question feel free to ask.
Priyanshu Srivastava I have thawed a new vial of cells. This time I centrifuged them for 2 minutes at 1000rpm during thawing and my cell pellet looks white and normal. When I sub-cultured them they looked grey and turned black during the second culturing. Is there any problem with freezing or the incubator? I have attached the microscopic image of the cells that I took with my phone.
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