I am working with human meniscus cells . For performing GAG and Collagen estimation from cultured cells in different time intervals like 2, 4, 6, 8 days , what is the best method of sampling ? Is it better taking a fraction of medium or a whole medium from culture wells? And how frequently should i change medium after taking samples for analysis?
I'd recommend taking the whole volume of medium for sampling as and when you feed the cultures (on a predetermined time course). that way you can concentrate the samples if you don't have sufficient total ECM proteins for robust detection. The amount of cells present will determine how long it takes to produce detectable amounts of ECM e.g. I leave 10,000 corneal fibroblasts for 7 days before assessing collagen production.
What are your end point assays?
Dimethylmethylene blue (DMMB) for GAG /sirius red for collagen? Immunos/ELISA? Mass Spectrometry?
Also, do you need to normalise these values to cell number?
Just to add to what Merlin has said, you can also perform Western blots on cell culture medium. I found an excellent collagen I antibody and was able to detect collagen in cell supernatant.
I grew 100,000 cardiac fibroblasts in 12 well dishes for 24 hr to 7 days in about 600 uL of medium. Since I was sampling the collagen from the supernatant I collected all of the medium and afterwards I counted the number of cells to be able to normalize to cell number/well. I added my running buffer directly to the medium and ran a normal Western blot.
Has Merlin Walter and Kristin Dawson, said different cell lines required different time points for sampling.
I use to treat primary human dermal fibroblast every 24 hrs with drug for 48 hrs seed in 96 well plate with 0.225 mL of medium with test substance. At 48th hrs, I use to collect 0.2 mL of supernatant for collagen estimation with Sircol dye binding assay from Biocolours.
I use only 48th hr sample for collagen estimation, Ascorbic acid and TGF beta showed dose dependent stimulation of collagen levels
Biocolours even sells GAG estimation kit.
What concentration of PEG in TRIS-HCl should be used for concentrating the culture medium for soluble collagen estimation?
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