Fresh thymocytes were resuspended in isolation buffer (PBS without cations, pH 7.2–7.4, containing 0.1% BSA and 2 mM EDTA) on ice. For CD4+ CD8+ T cell depletion, 5 × 107 thymocytes were incubated with 20 µg anti-CD4 (rat IgG2b clone GK1.5; BioXCell, West Lebanon, NH) and 37.5 µg anti-CD8a (rat IgG2a clone 53–6.72; BioXCell) in 5 ml isolation buffer for 20 min at 4°C with tilted rotation, centrifuged, resuspended in 5 ml isolation buffer, and incubated twice with 250 µl sheep anti-rat IgG Dynabeads (Thermo Fisher Scientific, Waltham, MA) for 30 min at 4°C with tilted rotation. After each incubation, the tube was placed in a magnet for 2 min, unbound DN T cell progenitors were combined in a new tube, centrifuged and RNA was extracted from the cell pellet with 1 ml TRIZOL® (Ambion, Carlsbad, CA ) as described below.
Anti cd8a rat igg2a clone 53 6.72
Manufactured by BioXCell
Anti-CD8a (rat IgG2a clone 53-6.72) is a monoclonal antibody that binds to the CD8a molecule, which is expressed on the surface of cytotoxic T cells.
Automatically generated - may contain errors
3 protocols using anti cd8a rat igg2a clone 53 6.72
1
Isolation of Double-Negative T Cell Progenitors
2
Isolation of Double Negative T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh thymocytes were resuspended in isolation buffer (PBS lacking cations, pH 7.2-7.4, containing 0.1% BSA, 2 mM EDTA and 1 mg/ml glucose) on ice. For T cell depletion, ~3-5x107 thymocytes were incubated with 20 μg anti-CD4 (rat IgG2b clone GK1.5, BioXCell) and 37.5 μg anti-CD8a (rat IgG2a clone 53-6.72; BioXCell) in 5 ml buffer for 20 min at 4°C with tilted rotation. After centrifugation, cells were resuspended in 5 ml buffer, and incubated with 250 μl sheep anti-rat IgG Dynabeads (Thermo Fisher Scientific) for 30 min at 4°C with tilted rotation. The tube was placed in a magnet for 2 min, unbound cells were centrifuged and resuspended in 250 μl Dynabeads for a second 30 min incubation at 4°C. After Dynabeads removal, unbound DN T cells were centrifuged, counted and RNA was extracted from the cell pellet with 1 ml TRIZOL (Ambion) as described below.
3
Thymocyte Isolation and T Cell Depletion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh thymocytes were resuspended in isolation buffer (PBS lacking cations, pH 7.2-7.4, containing 0.1% BSA, 2 mM EDTA and 1 mg/ml glucose) on ice. For T cell depletion, ~3-5x10 7 thymocytes were incubated with 20 µg anti-CD4 (rat IgG2b clone GK1.5, BioXCell) and 37.5 µg anti-CD8a (rat IgG2a clone 53-6.72; BioXCell) in 5 ml buffer for 20 min at 4°C with tilted rotation. After centrifugation, cells were resuspended in 5 ml buffer, and incubated with 250 µl sheep anti-rat IgG Dynabeads (Thermo Fisher Scientific) for 30 min at 4°C with tilted rotation. The tube was placed in a magnet for 2 min, unbound cells were centrifuged and resuspended in 250 µl Dynabeads for a second 30 min incubation at 4°C. After Dynabeads removal, unbound cells were centrifuged, counted and RNA was extracted from the cell pellet with 1 ml TRIZOL (Ambion) as described below.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!