I am currently inducing human pluripotent stem cells towards the hematopoietic lineage and assessing the functional capacity of the progenitors by performing colony based assay on MethoCult. I see some CFUs in the dish but I am not able to image them properly despite of using a high resolution camera.
Hi Kaja: Any inverted microscope equipped with 10X eyepiece objective and 2X, 4X, and 10X planar objectives and blue filter is recommended. Please see the following technical manual:
https://www.stemcell.com/media/files/manual/MA28404-Human_Colony_Forming_Unit_Assays_Using_MethoCult.pdf
I hope it helps, cheers and good luck with your experiment!!! Rafael.
Hi Kaja,
As Rafael Flores-Obando said an inverted microscope should be more than sufficient. Focusing may be difficult as the colonies exist in multiple fields of view. It is important to ensure the formulation you are using is designed for proper growth of colonies, MethoCult SF H4636 is designed for colony assays of iPSC-derived hematopoietic progenitor cells.
Additionally if you are having issues imaging colonies at the edges of wells, meniscus-free cultureware can be used to avoid shadows or crowding of colonies at well edges. More info here:
https://www.stemcell.com/media/files/brochure/BR28017-SmartDish_STEMgrid6_Meniscus_Free_Cultureware_More_Accurate_Counting_Hematopoietic_Colonies.pdf?utm_source=researchgate
Let me know if you have any questions! Good luck :)
Thank you so much Rafael Flores-Obando Amanda Fentiman for your suggestions. I am using MethoCult H4434 CLASSIC from StemCell Technologies and an old Zeiss upright microscope for imaging my colonies. I am able to observe colonies in the dish by adjusting the light beneath the dish but I am not sure about their category. Please suggest me with a microscope model number that I can use. Here are some of the images taken using the Zeiss microscope.
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