Biotin Rat Anti-Mouse CD45

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	1 μl biotin CD45

Upstream 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
Protocol tips
1 μl biotin CD45

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).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Flow cytometry Anti-bodies Mouse - CD45 using Biotin Rat Anti-Mouse CD45 from BD Biosciences.

Manufacturer protocol

Download the product protocol from BD Biosciences for Biotin Rat Anti-Mouse CD45 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Flow cytometry Anti-bodies Mouse - CD45 using Biotin Rat Anti-Mouse CD45 from BD Biosciences. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.