Preparation And Storage
Recommended Assay Procedures
For flow cytometric applications, a three step labeling procedure is recommended for amplifying signal. Suggested protocol for 3-step staining using Lysed Whole Blood method:
1. Incubate 100µl whole blood with primary (unconjugated) antibody for 20-30 minutes in the dark at room temperature.
2. Add 2 mls of 1X Lysing Buffer (Cat. No. 555899/349202) and incubate for 10-15 minutes in the dark. Centrifuge and aspirate.
3. Wash once with PBS/0.1% sodium azide/1% heat-inactivated fetal bovine serum (PBS-FBS). Centrifuge and aspirate.
4. Add biotinylated goat anti-rat Ig's and incubate for 20-30 minutes in the dark at room temperature.
5. Wash once with PBS-FBS. Centrifuge and aspirate.
6. Add SAV-PE (Cat. No. 554061) and incubate for 20-30 minutes in the dark at room temperature.
7. Wash once with PBS-FBS. Centrifuge and aspirate. Resuspend in 0.5 ml of PBS-FBS and analyze by flow cytometry.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
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The 3F9 monoclonal antibody specifically binds to CD210a, which is also known as, Interleukin-10 Receptor subunit alpha (IL-10R subunit alpha/IL-10Rα/IL10RA), or Interleukin-10 receptor subunit 1 (IL-10R1). CD210a is a 90-110 kDa type I transmembrane glycoprotein that belongs to the type II cytokine receptor family. CD210a combines with IL-10 Receptor subunit beta (IL-10Rβ/IL10RB/CDw210b) to form the IL-10 Receptor complex (IL-10Rα/IL-10Rβ) that can bind IL-10 and transduce signals intracellularly through the JAK/STAT pathway. IL-10 can suppress antigen presentation and the expression of proinflammatory type-1 immune responses while promoting type-2 immune responses. CD210a is expressed on T cells, B cells, NK cells, monocyte, macrophages and dendritic cells. Clone 3F9 is specific for human CD210a and its binding to the receptor can be blocked by recombinant human IL-10 (rhIL-10) protein.
Development References (4)
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Callard R, Gearing A. Callard R, Gearing A. The Cytokine Facts Book. San Diego: Academic Press; 1994.
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Carson WE, Lindemann MJ, Baiocchi R, et al. The functional characterization of interleukin-10 receptor expression on human natural killer cells. Blood. 1995; 85(12):3577-3585. (Biology). View Reference
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Liu Y, Wei SH, Ho AS, de Waal Malefyt R, Moore KW. Expression cloning and characterization of a human IL-10 receptor. J Immunol. 1995; 152(4):1821-1829. (Biology). View Reference
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Liu Y, de Waal Malefyt R, Briere F, et al. The EBV IL-10 homologue is a selective agonist with impaired binding to the IL-10 receptor.. J Immunol. 1997; 158(2):604-13. (Biology). View Reference
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