I already follow some troubleshoot in AGT book and some of cytogenetic's journal. I have tried to change the concentration of trypsin and giemsa but I still dont get the band (see the picture)
These are what I have already tried:
1. using 0.5% trypsin (10-60s) and 10% giemsa (8 min - 15 min)
2. 1% trypsin (10-60s) and 10% giemsa (8 min - 15 min)
3. 2.5% trypsin (10-60s) and 10% giemsa (8 min - 15 min)
4. 0.5% trypsin (10-60s) , FBS and 10% giemsa (8 min - 15 min)
5. 1% hot trypsin (10-60s) and 10% giemsa (8 min - 15 min)
6. freeze the slide and trypsinzed using 1% trypsin (10-60s) and staining with 10% giemsa (8 min - 15 min)
7. mixed 1% trypsin and 10% giemsa in one jar (8 min - 15 min)
I diluted the trypsin using PBS with pH 7 and diluted the giemsa using PB with pH 6.
The trypsin is from Gibco (1:250) and the giemsa is from Merck.
is it possible because my slide is too old? so the trypsin can not digest the histones and make the band dont appear?
Please could someone help me to solve this problem?
Getting good banding requires a balance between both trypsin and stain times. Your stain times seem too long and trypsin times may be too short.
How old are the slides?
Oke, thanks for your suggestion.
my slides maybe around 1-3 months. all my slides less than 6 months.
Trypsin timing will vary from different tissue, the metaphases are from which tissue ?... also the metaphases seems to have not divided completely.
Rina, they should be fine when "around 1-3 months."
Debarshi, Given that these are metaphase chromosomes, what do you mean that they have not divided completely?
please share with me the metaphases are from blood, amniotic fluid, cvs or bone marrow I shall then explain further which will give more clear picture
Debarshi Sanyal The metaphases are from peripheral blood. We use around 7 drops buffy coat and mix it with 8 ml MEM + 100ul PHA + 50 ul Antibiotic + 100 ul Glutamine.
To mention, MEM is not very suited media for Blood/Buffy Coat ( Buffy Coat are from cell lines or extracted from blood by phycol please share this )
in addition the antibiotic I feel in hindering the division rate and the reason of poor quality metaphase ( the metaphases look bi armed )
As you have mentioned(MEM) there is lack of few essential growth factors which is required for good quality metaphases , hence even after a good harvest the histones and proteins are still present, which the current trypsin time is not able to digest and hence the bands are unclear. Lastly due to lack of growth factors and due to antibiotics the division not complete and its getting arrested by colchicine.
Please follow this
RPMI 1640 ( comes with antibiotics and L-Glutamine) if your choice is for a conventional media + FBS US origin ( HEAT INACTIVATED ) (10%) FBS
You can also try for PB MAX invitrogen which is ready to use media.
Next
8ML RPMI+ 2ml 10%FBS
100-150 mcl of buffy coat
200 mcl PHA ( M FORM)
FOLLOW your regular harvest protocol and prepare slides,
bake the slides at 60 degrees over nite before G Banding
Try this you will be a marked improvement in quality of metaphases as well a very high quality G Bands and later try for High resolution chromosomes and G Band
Hope this will be useful.
Rina Susanti Chen, Can you let me know where are you working which institute.
Thank you for your help Debarshi Sanyal
Actually, we use both MEM and RPMI for one sample. We dont use phycol to get the buffy coat but we just centrifuge the sample in 800xg, 10 min.
If it is because the poor quality of metaphase, I still can found the long chromosome without bi armed.
does it possible the trouble is in the trypsin? How can I know the trypsin work well in the chromosome? or maybe I need to dilute the trypsin using NaCl not PBS?
{ 2ml 10%FBS }--> What do you mean 2 ml 10%FBS?
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