I want to isolate CD8+ T cells from patient tumor and mouse tumor. Nowadays I just use collagenase type I 0.2% and incubate 45 min - 1hour --> Lymphoprep --> MACS isolation. However, my mixture after lysis is so sticky that I want to add DNase type IV and Hyaluronidase type V (the attached file).
1. Anyone has experience about this enzyme mixture? Please recommend the concentration and producers!
2. I'm still confused about Kunitz unit and U. For example Deoxyribonuclease I from bovine pancreas Type IV, lyophilized powder, ≥2,000 Kunitz units/mg protein (Sigma Aldrich), how can convert into U/mg.
Thank you.
Hi, for the preparation of mouse tumour samples for FACS we incubate with Collagenase D (2mg/ml) and DNAse I (100ug/ml), both purchased from Roche, for 1.5-2h @ 37°C. Good luck
@Digdem, I cannot use directly FACS sorting because in tumor there's just 3-5% CD8. And we must dissociate the tumor before MACS/FACS sorting.
@Tommaso Torcellan: Thank you very much.. I used to consider DNAse I from Roche. However they just sell the DNase 100mg (cat 10104159001). If you dilute 100g in 200ul, the final concentration is 1000 U/mL, whereas my desired working concentration is 2U/mL. On the other hand DNase I type IV from Sigma (D5025) has much higher activity 15.000 U (powder).
Finally, I decided to use below enzymes (all purchased from Sigma), 37oC,1-2 hours
- Collagenase I 0.2% (type I to obtain lymphocytes, type IV to obtain myeloid cells)
- DNase type IV 30U/ml
- Hyaluronidase type V 0.01%
Many thanks.
Collagenase type IV (1mg/ml) + DNAseI (20 ug/ml) ; both from Worthington. I use this mixture for mouse tumors. If the tumor is finely minced 30-60 min is enough.
You don't want to let the cells too long in the dissociation medium as it will damaged the cells but also the extracellular markers you want to analyse by FACS.
Additional question: what volume of digestion buffer (with Collagenase, DNAse, etc) do you use per tumour? Do you scale according to tumour weight?
Hi Pedro,
Mouse tumor : 5mL enzyme mixture for 1 tumor 1cm3
Human tumor: it depends on patients so I just estimate the necessary enzyme..We usually freeze 10mL/tubes, so after thawing I will use 5ml - 10mL -15mL-20mL per tumor.
@Bertrand Allard : you are right, it's really hard to confirm the purity of CD8+ cells. Cells populations usually clearly separates into 2 groups but enzymes may damage this marker.
Thank you Hanh!
I have read that both HBSS or RPMI can be used to dilute the enzymes - which buffer do you use?
You're welcome, Pedro!
As I read a document from NIH, they recommend that 10X triple enzyme should be dissolved in HBSS (so tumor will also be soaked in HBSS during dissociation). So now I'm using HBSS for all steps: stock, dillute, washing...
However, I know another lab is using RPMI to make 1X stock but I'm not sure about how it affect on the result because of different purposes.
Hope that helps.
Hi to all,
i have this product :
-Hyaluronidase from bovine testes Type IV-S, lyophilized powder (essentially salt-free), 750-3000 units/mg solid
-Collagenase from Clostridium histolyticum purified by chromatography, ≥1 FALGPA units/mg solid, ≥250 CDU/mg solid (CDU = collagen digestion units), lyophilized powder
Synonym: Clostridiopeptidase A
I have to prepare a mixture for a digestion buffer and the protocol that i follow says me that I have to use it at a 1X concetration.
Somebody of you suggest me how I reconstituition this product.
thank for the helping
What about Liberase? Has anyone every used it? What kind of liberase you have used and also in what concentrations?
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