Hi, I’m doing transwell migration with B16F10 cells recently. But I ran into a problem after staining with 1% crystal violet, some dirty print showed in background (shown as figures). What could it be? How could I clean it?
I used a 8.0 μm insert in 24 well plate and my steps are briefly as followed:
1. Seed 4*10^4 B16F10 cells with 100 ul serum free medium to the upper compartment of the insert.
2. Add 0.6ml DMEM supplemented with 10% FBS to the lower chamber as attractant.
3. After Incubation for 24 hours, take the insert out carefully. Remove cells in the upper compartment of the insert by gently wiping the upper side of the membrane with a cotton swab.
4. Fix the cells on the lower side of the insert membrane with 100% methanol for 10 min, followed by staining with 1% crystal violet in methanol for additional 20 min.
5. Wash the insert in PBS for several seconds to remove excess dye.
6. Remove excess dye and cells in the upper compartment of the insert by gently wiping the upper side of the membrane with a cotton swab.
7. Observe under a microscope.
BTW, when I did transwell migration with HOS cells (2*10^4 cells/well), the background was clean.
Have you tried some negative controls?
1. Stain an unused insert to see if it's a bad batch of inserts or your crystal violet solution.
-to tell the difference , make new stain and filter with 0.2 micron filter.
-try a different stain
2. Stain culture inserts after incubation with your culture medium components: something in DMEM or your FBS Is a adsorbing omto the insert.
if those are clean then then I would suspect deposition/secretion of material by your cell line.
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