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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
| Skin tissue was incubated for 90 min at 37°C in dispase (2.5mg/ml) followed by the separation of epidermis and dermis. Epidermal sheets were subsequently incubated for 30 min in trypsin/EDTA and remaining skin tissue was chopped into small fragments and incubated for 30 min at 37°C in collagenase type 3 (3 mg/ml). |
For recovery of T cells from the lung, mice were injected i.v. with 3 μg of Alexa Fluor 700-conjugated antibody to CD3 10 min prior to sacrifice, and mice were then perfused before the collection of lung tissue that was digested for 90 min in collagenase type 3 (3 mg/ml). For intracellular staining, cells were fixed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) before staining with intracellular antibodies. |
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| Upstream tips |
| Skin tissue was incubated for 90 min at 37°C in dispase (2.5mg/ml) followed by the separation of epidermis and dermis. Epidermal sheets were subsequently incubated for 30 min in trypsin/EDTA and remaining skin tissue was chopped into small fragments and incubated for 30 min at 37°C in collagenase type 3 (3 mg/ml). |
| Protocol tips |
| For recovery of T cells from the lung, mice were injected i.v. with 3 μg of Alexa Fluor 700-conjugated antibody to CD3 10 min prior to sacrifice, and mice were then perfused before the collection of lung tissue that was digested for 90 min in collagenase type 3 (3 mg/ml). For intracellular staining, cells were fixed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) before staining with intracellular antibodies. |
| Upstream tips |
Protocol tips |
Downstream tips |
| Single-cell suspensions were incubated with a Fc receptor block (1 μg/1 × 106 cells; BD Bioscience, San Diego, CA) to reduce nonspecific anti-body binding. |
Dead cells were excluded using a Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen) or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO). |
Flow cytometry was performed using BD LSR II and BD FACS Aria III flow cytometers (BD Bioscience), and data were analyzed with FlowJo software (TreeStar, Ashland, OR). |
| Upstream tips |
| Single-cell suspensions were incubated with a Fc receptor block (1 μg/1 × 106 cells; BD Bioscience, San Diego, CA) to reduce nonspecific anti-body binding. |
| Protocol tips |
| Dead cells were excluded using a Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen) or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO). |
| Downstream tips |
| Flow cytometry was performed using BD LSR II and BD FACS Aria III flow cytometers (BD Bioscience), and data were analyzed with FlowJo software (TreeStar, Ashland, OR). |
| Upstream tips |
Protocol tips |
Downstream tips |
| For staining of mouse lymph nodes, cells were prepared by rapidly passing the whole node through a 40 um mesh, then stained using short incubation times (10 min on ice), as described (Sharma et al., 2013). |
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| Upstream tips |
| For staining of mouse lymph nodes, cells were prepared by rapidly passing the whole node through a 40 um mesh, then stained using short incubation times (10 min on ice), as described (Sharma et al., 2013). |
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