Alexa Fluor® 647 Rat anti-Mouse CD34

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 10 μl cell suspension from the 2 ml sort sample in 500 μl media and the following volumes of antibody to separate 5 ml FACS tubes for single color controls. 4 μl CD34/Alexa Fluor 647	Keep on ice until ready for sorting on a BD Biosciences FACSAria cell sorter

Protocol tips
Add 10 μl cell suspension from the 2 ml sort sample in 500 μl media and the following volumes of antibody to separate 5 ml FACS tubes for single color controls. 4 μl CD34/Alexa Fluor 647
Downstream tips
Keep on ice until ready for sorting on a BD Biosciences FACSAria cell sorter

Publication protocol

"1. Add 10 μl cell suspension from the 2 ml sort sample in 500 μl media and the following volumes of antibody to separate 5 ml FACS tubes for single color controls:
a. No antibody (unstained control)
b. 1 μl FxCycle Violet stain (DAPI)
c. 1 μ a7-integrin/PE or FITC
d. 4 μl CD34/Alexa Fluor 647
e. 1 μl biotin CD45
2. Add the following volumes of primary antibodies to together the original 2 ml sort samples:
a. 5 μl biotin CD45
b. 10 μl biotin CD11b
c. 10 μl biotin CD31
d. 10 μl biotin Ly-6A/E-Sca1
3. Gently vortex samples and incubate on ice for 20 min.
4. Add 10 ml chilled media to sort samples or 3 ml FACS buffer to single color controls.
5. Centrifuge samples at 300 x g for 5 min at 4 °C.
6. Aspirate supernatant, resuspend sort samples in 1.5 ml media or single color controls in 500 μl FACS buffer.
7. Add 1 μl streptavidin APC-Cy7 to the biotin CD45 single color control and incubate on ice for 20 min.
8. Add the following secondary antibodies to sort samples (microbeads first, then remaining antibodies):
a. 150 μl streptavidin microbeads
b. 10 μl a7-integrin/PE or FITC
c. 30 μl CD34/Alexa Fluor 647
d. 10 μl streptavidin APC-Cy7
9. Gently vortex samples and incubate on ice for 20 min.
10. Add 10 ml chilled media to sort samples or 3 ml FACS buffer to the biotin CD45 single color control.
11. Centrifuge samples at 300 x g for 5 min at 4 °C.
12. Pre-load LS columns (one for each sample) for depletion with 3 ml media.
13. Aspirate supernatant, resuspend sort samples in 1 ml media or the biotin CD45 single color control in 500 μl FACS buffer.
14. Load each sort sample onto an LS column placed on a MACS magnetic separator, washing the original centrifuge tube 2 times with 3–4 ml media (keeping samples on ice as much as possible).
15. Centrifuge samples at 300 x g for 5 min at 4 °C.
16. Aspirate supernatant, resuspend sort samples in 2 ml FACS buffer transferred to 5 ml FACS tubes and add 2 μl FxCycle Violet stain (DAPI) to each sample.
17. Keep on ice until ready for sorting on a BD Biosciences FACSAria cell sorter (Figure 3).
"

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Manufacturer protocol

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