Alexa Fluor® 647 anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody

Product Details

Verified Reactivity

Mouse

Antibody Type

Monoclonal

Host Species

Rat

Immunogen

Raised against granulocytes of mouse origin

Formulation

Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.

Preparation

The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 647 under optimal conditions.

Concentration

0.5 mg/ml

Storage & Handling

The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.

Application

FC - Quality tested
SB - Community verified

Recommended Usage

Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is ≤ 0.25 µg per 10⁶ cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application.

* Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633nm / 635nm.

Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.

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Excitation Laser

Red Laser (633 nm)

Application Notes

Clone RB6-8C5 binds with high affinity to mouse Ly-6G molecules and to a lower extent to Ly-6C¹⁹. Clone RB6-8C5 impairs the binding of anti-mouse Ly-6G clone 1A8¹⁹. However, clone RB6-8C5 is able to stain in the presence of anti-mouse Ly-6C clone HK1.4²⁰.

The RB6-8C5 antibody has been used to identify peripheral blood neutrophils and deplete granulocytes in vivo. Additional reported applications (for relevant formats of this clone) include: in vitro complement-mediated cytotoxicity², in vivo depletion^3-5,9, immunoprecipitation¹, immunohistochemical staining⁶ (including paraffin-embedded sections^9,16,33-35, acetone-fixed frozen sections¹¹ and zinc-fixed sections¹⁵), and Western blotting⁷. RB6-8C5 is not suitable for depletion of hepatic myeloid derived suppressor cells (MDSCs)²⁰.

Special Note: For in vivo studies or highly sensitive assays, we recommend Ultra-LEAF™ purified antibody (Cat. No. 108436).

Additional Product Notes

This product has been verified for IHC-F (Immunohistochemistry - frozen tissue sections) on the NanoString GeoMx® Digital Spatial Profiler. The GeoMx® enables researchers to perform spatial analysis of protein and RNA targets in FFPE and fresh frozen human and mouse samples. For more information about our spatial biology products and the GeoMx® platform, please visit our spatial biology page.

Application References

(PubMed link indicates BioLegend citation)

1. Fleming TJ, et al. 1993. J. Immunol. 151:2399. (IP)
2. Brummer E, et al. 1984. J. Leukocyte Biol. 36:505. (CMCD)
3. Stoppacciaro A, et al. 1993. J. Exp. Med. 178:151. (Deplete)
4. Tumpey TM, et al. 1996. J. Virol. 70:898. (Deplete)
5. Czuprynski CJ, et al. 1994. J. Immunol. 152:1836. (Deplete)
6. Nitta H, et al. 1997. Cell Vision 4:73. (IHC)
7. Jutila MA, et al. 1988. Eur. J. Immunol. 18:1819. (WB)
8. Engwerda CR, et al. 2004. Am. J. Pathol. 165:2123.
9. Brown CR, et al. 2004. Infect. Immun. 72:4956. (Deplete, IHC)
10. Andoniou CE, et al. 2005. Nature Immunology 6:1011. (FC) PubMed
11. Li M, et al. 2006. P. Natl. Acad. Sci USA 103:11736. (IHC)
12. Dzhagalov I, et al. 2007. Blood 109:1620. (FC) PubMed
13. Fazilleau N, et al. 2007. Nature Immunol. 8:753. (FC) PubMed
14. Heuser M, et al. 2007. Blood 110:1639. (FC) PubMed
15. Wang T, et al. 2007. Infect. Immun. 75:1144. (IHC)
16. Bosio CM, et al. 2007. J. Immunol. 178:4538. (IHC)
17. Boehme SA, et al. 2009. Int. Immunol. 21:81. (IHC)
18. Piao Y, et al. 2012. Neuro Oncol. 14:1379. PubMed
19. Ribechini E, et al. 2009. Eur. J. Immunol. 39:3538.
20. Ma C, et al. 2012. J. Leukoc. Biol. 92:1199.
21. Li J, et al. 2012. Arthritis Rheum. 64:1098. PubMed
22. Fan Q, et al. 2014. Cancer Res. 74:471. PubMed
23. Korrer MJ, et al. 2014. PLoS One. 9:91370. PubMed
24. Morshed M, et al. 2014. J Immunol. 192:5314. PubMed
25. Collins C, et al. 2014. PNAS. 111:9899. PubMed
26. Madireddi S, et al. 2014. J Exp Med. 211:1433. PubMed
27. Bianchi G, et al. 2014. Cell Death Dis. 5:1135. PubMed
28. Guo H, et al. 2014. J Leukoc Biol. 96:419. PubMed
29. Roderick JE, et al. 2014. PNAS. 111:14436. PubMed
30. Distel E, et al. 2014. Circ Res. 115:759. PubMed
31. Iwai H, et al. 2015. Tuberculosis. 95:246. PubMed
32. Charmsaz S, et al. 2015. PLoS One. 10:130692. PubMed
33. Whiteland J, et al. 1994 J Histochem Cytochem 43:3 (IHC-P)
34. Brown C, et al. 2003 J Immunology 171:2 (IHC-P)
35. Obregon-Henao A, et al. PLoS One 8:11 (IHC-P)

Product Citations

1. Madrid-Paulino E, et al. 2022. J Leukoc Biol. 112:475. PubMed
2. Barsheshet Y, et al. 2022. Int J Mol Sci. 23:. PubMed
3. Kang YA, et al. 2023. J Exp Med. 220:. PubMed
4. Doloff JC, et al. 2023. Sci Adv. 9:eade9488. PubMed
5. Tan L, et al. 2022. Biochem Biophys Rep. 32:101351. PubMed
6. Tarban N, et al. 2022. Cells. 11:. PubMed
7. Wang J, et al. 2012. Blood. 120:1489. PubMed
8. Ni J, et al. 2022. Mol Med. 28:65. PubMed
9. Demars A, et al. 2021. PLoS Pathog. 17:e1009887. PubMed
10. Shaw O, Harper J 2011. Biochem Biophys Res Commun. 416:266. PubMed
11. Zhang H, et al. 2017. Leukemia. 10.1128/mBio.00226-17. PubMed
12. Koliaraki V et al. 2019. Cell reports. 26(3):536-545 . PubMed
13. Bittner–Eddy PD, et al. 2017. Front Immunol. 1.304166667. PubMed
14. Chen G, et al. 2015. Sci Rep. 5: 14780. PubMed
15. Meraz I, et al. 2014. PLoS One. 9:94703. PubMed
16. Wheeler R, Gull E 2015. Nat Commun. 6: 8964. PubMed
17. Bouchery T, et al. 2020. Cell Host & Microbe. 27(2):277-289. PubMed
18. Biburger M, Nimmerjahn F 2012. Immunol Lett. 143:53. PubMed
19. Tummers B, et al. 2020. Immunity. 52(6):994-1006.e8. PubMed
20. Kim K, et al. 2013. J Vis Exp. 74: 50329. PubMed
21. Budai Z, et al. 2021. Cells. 10:. PubMed
22. Hahm E, et al. 2016. Nat Med. 23:100-106. PubMed
23. Hendrikx S et al. 2019. Cell reports. 26(5):1227-1241 . PubMed
24. Rossnagl S, et al. 2016. PLoS Biol. 14: 1002562. PubMed
25. Al-Zaeed N, et al. 2021. Cell Death Dis. 12:611. PubMed

RRID

AB_493481 (BioLegend Cat. No. 108420)
AB_493481 (BioLegend Cat. No. 108418)

Antigen Details

Structure

21-25 kD

Distribution

Granulocytes, monocytes

Cell Type

Granulocytes, Monocytes, Neutrophils

Biology Area

Immunology, Innate Immunity

Antigen References

1. Fleming TJ, et al. 1993. J. Immunol. 151:2399.
2. Jutila MA, et al. 1988. Eur. J. Immunol. 18:1819.
3. Goni O, et al. 2002. Int. Immunol. 14:1125.

Gene ID

17067 View all products for this Gene ID 546644 View all products for this Gene ID

UniProt

View information about Ly-6G Ly-6C on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Protocols

• Cell Surface Flow Cytometry Staining Protocol

Related Pages & Pathways

Related FAQs

If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?

It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.

Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?

Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.

Are other fluorophores compatible with IBEX?

Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.

The same antibody works in one tissue type but not another. What is happening?

Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.

How can I be sure the staining I’m seeing in my tissue is real?

In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.

Other Formats

Certificate of Analysis