Pacific Blue™ anti-mouse Ly-6A/E (Sca-1) Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Transfer 10 μl of the cell suspension to a 5-ml FACS tube containing 190 μl WM and leave on ice. This is the unstained control sample.	Add 190 μl WM into each tube and 10 μl of the cell suspension. Add PI so that the final concentration is 0.3 μg/ml, add 0.25 μl Ab

Upstream tips
Transfer 10 μl of the cell suspension to a 5-ml FACS tube containing 190 μl WM and leave on ice. This is the unstained control sample.
Protocol tips
Add 190 μl WM into each tube and 10 μl of the cell suspension. Add PI so that the final concentration is 0.3 μg/ml, add 0.25 μl Ab

Publication protocol

"1. Transfer 10 μl of the cell suspension to a 5-ml FACS tube containing 190 μl WM and leave on ice. This is the unstained control sample.
2. Label nine 2-ml tubes to prepare for the single channel and fluorescence minus one (FMO) staining controls for each individual channel, APC, Pacific Blue and PE-Cy7, and antibody isotype controls. Add 190 μl WM into each tube and 10 μl of the cell suspension. Add PI so that the final concentration is 0.3 μg/ml, add 0.25 μl CD31-APC and CD45-APC, Sca1-Pacific Blue or VCAM1-Biotin antibodies to the appropriately labeled tubes. If the concentration of isotype control antibody differs from a given specific antibody, calculate the volume before applying so that the same amount of isotype control antibody is used.
3. Transfer the rest of the cell suspension (about 500 μl) into a labeled 2-ml tube. For cells from each mouse, add 5 μl of each of the following antibodies: CD31-APC, CD45-APC, Sca1-Pacific Blue and VCAM1-Biotin. CRITICAL STEP: Assuming a typical yield, all antibodies are used at about 1 μg per 107 cells except for VCAM1-Biotin which is used at about 2.5 μg per 107 cells. Titration of antibodies is usually not required when using reagents suggested by this protocol. However, optimization may be necessary for antibodies from other vendors.
4. Place all staining samples on a nutating rocker at 4°C and incubate for 40 min.
5. Fill all tubes to 2 ml with WM. Gently invert the tubes a few times to mix. Centrifuge at 250g with a fixed angle rotor for 5 min at 4°C.
6. Aspirate supernatant completely. Resuspend cells in control tubes in 200 μl WM and the sort sample in 500 μl WM. CRITICAL STEP: Be careful not to aspirate cells. It helps to aspirate with low-volume pipet tips with narrow openings, e.g. gel-loading tips or 1–10 μl tips.
7. Add 0.5 μl PE-Cy7 Streptavidin into the control tubes 3–5 and 7–9.
8. Add 5 μl PE-Cy7 Streptavidin and PI to a final concentration to 0.3 μg/ml into the sort sample. Place all tubes on a nutating rocker at 4°C and incubate for 20 min.
9. Transfer the cells in control tubes 1, 2 and 4 to labeled 5-ml FACS tubes. Leave the tubes on ice and protect from light.
10. Fill all tubes to 2 ml with WM. Gently invert the tubes a few times to mix. Centrifuge at 250g with a fixed angle rotor for 5 min at 4°C. Aspirate supernatant completely. CRITICAL STEP: Be careful not to aspirate cells. It helps to aspirate with low-volume pipet tips with narrow openings, e.g. gel-loading tips or 1–10 μl tips.
11. Resuspend cells in control tubes in 200 μl WM and transfer to labeled 5-ml FACS tubes.
12. Resuspend cells in the sort sample in 500 μl WM. Transfer to the strainer cap attached to a 5-ml FACS tube. Gently tap the tube on the bench to facilitate flow through.
13. Rinse the 2-ml tube used to stain the sort sample with another 500 μl WM and pass through the same strainer cap. Use a 20–200 μl pipet to collect any remaining liquid in the strainer cap from the underside. Add another 1 ml of WM to the sort sample and mix by gentle vortex. CRITICAL STEP: It is best to move immediately to cell sorting for the highest yield and viability."

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Manufacturer protocol

Download the product protocol from BioLegend for Pacific Blue™ anti-mouse Ly-6A/E (Sca-1) Antibody below.

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