CR2 Antibody - PE Conjugated (OASA00425)

Catalog No: OASA00425 (Formerly GWB-Q00864)

Size:100T

Staining of canine peripheral blood lymphocytes with Mouse anti Canine CD21:RPE

Staining of canine peripheral blood lymphocytes with Mouse anti Canine CD21

Staining of canine peripheral blood lymphocytes with Mouse anti Canine CD21:Alexa Fluor 647

Product Info

• Predicted Species Reactivity: Dog
• Product Format: Lyophilized. Phosphate buffered saline
• Clonality: Monoclonal
• Clone: CA2.1D6
• Isotype: IgG1
• Host: Mouse
• Conjugation: R-PE
• Application: FC
  Preparation: Purified IgG prepared by affinity chromatography on Protein G
  Preservative Stabilisers: 0.09% - Sodium Azide
  1% - Bovine Serum Albumin
  Buffer Solutions: Phosphate buffered saline pH7.4
• Reconstitution and Storage: 2°C to 8°C
• Purification: Protein G Purified
• Predicted Homology Based on Immunogen Sequence: Dog
• Specificity: OASA00425 recognizes CD21, a cell surface antigen expressed by canine B lymphocytes.
• Application Info: Flow Cytometry: Use 10⁶ ul of the suggested working dilution to label 106 cells or cells or 100 ul whole food.
• Application Data: Application #1: Staining of canine peripheral blood lymphocytes with Mouse anti Canine CD21:RPE
  Application #2: Staining of canine peripheral blood lymphocytes with Mouse anti Canine CD21
  Application #3: Staining of canine peripheral blood lymphocytes with Mouse anti Canine CD21:Alexa Fluor 647
• Protocol Information: Citation: 1: Huang YC, Hung SW, Jan TR, Liao KW, Cheng CH, Wang YS, Chu RM. CD5-lowexpression lymphocytes in canine peripheral blood show characteristics of naturalkiller cells. J Leukoc Biol. 2008 Dec;84(6):1501-10. Epub 2008 Aug 15. PubMedPMID: 18708592.
  Species: Beagles
  Experiment Name: 1. Flow cytometry analysis2. Detection of intracellular IFN-
  Experiment Background: NK cell markers and receptors have been discovered in many mammalian species, such as humans, mice, rats, pigs, and cows. However, there is still a lack of information concerning NK cell markers or receptors in canines. Yi-Chun et al. have discovered that canine CD5-low density (CD5lo) cells in PBL are closely associated with NK cell characteristics. CD5lo cells comprised 14.9 ± 6.68% of the total PBL. A high proportion of the CD5lo cell population expressed CD3 (96.6%), CD8α (77.7%), CD8β (53%), α/β TCR (83%), and CD11/18 (80%), but the expression of / TCR (6.5%), CD4 (10.6%), and CD21 (2.4%) was low. CD5lo cells were larger than CD5-high density (CD5hi) cells. Light and electron microscopy revealed numerous large cytoplasmic granules in CD5lo cells, especially after IL-2 stimulation, which was in contrast to CD5hi, in which intracytoplasmic granules were not frequently seen. After IL-2 stimulation, CD5lo cells had significantly stronger NK cytotoxicity than CD5hi cells. CD5lo cells had much higher mRNA levels for NKG2D, CD16, CD94, CD160, perforin, and granzyme than CD5hi. Following IL-2 stimulation, CD5lo cells had significantly higher mRNA levels of NKp30, NKp44, CD16, and CD94 than CD5hi cells. In addition, IL-2-stimulated, CD5lo-depleted PBL showed a loss of NK cytotoxicity. CD5lo cells also showed significantly lower antigen-specific cytotoxic T cell activity as compared with CD5hi cells. Taken together, the CD5lo subset in canine PBL is closely related to canine NK cells, and CD5lo can be used as a phenotypic marker for an IL-2-dependent canine NK cell enrichment.
  Experimental Steps: 1. Flow cytometry analysis: Cells were stained as described previously [25]. Briefly, for direct immunofluorescence analysis, cells were incubated with FITC-conjugated isotype control or FITC-conjugated rat anti-canine CD5 mAb (Oxford, UK). For indirect immunofluorescence analysis, cells were incubated with isotype control or specific mAb raised against CD3, CD4, CD8α, CD8β, CD21, α/β TCR, / TCR, CD14, MHC class I, MHC class II, or CD11/18. Cells were washed and further stained with PE-conjugated goat anti-mouse IgG (Becton Dickinson, San Jose, CA, USA) for 30 min. Finally, all cells were washed and suspended in FACS buffer [2% FBS and 0.02% sodium azide in PBS (pH 7.4)]. The surface immunofluorescence of 104 viable cells was measured with a FACSCaliburTM flow cytometer (Becton Dickinson). Data were analyzed using CellQuest software (Becton Dickinson).2. Detection of intracellular IFN-: Canine PBMCs were analyzed for intracellular IFN- by a flow cytometer. Briefly, PBMCs were incubated in recombinant canine (rc)IL-12 (5 ng/ml; R&D System, Minneapolis, MN, USA) and were collected after 72 h. The cells were then processed for cell surface staining using FITC-conjugated anticanine CD5 mAb. Intracellular staining was performed using anti-IFN--PE (CC302; Oxford, UK) with a BD Cytofix/Cytoperm Plus kit and GolgiStop (Becton Dickinson), according to the manufacturer’s instructions. Cells (10,000)/sample were analyzed in a FACSCaliburTM flow cytometer.
  Other Reagents Used: RPMI-1640, penicillin, streptomycin, 2-ME, glutamine
  Number Of Protocols: 2

Target Info

• Reference: 1. Cobbold, S. P. and Metcalf, S. (1994) Monoclonal antibodies that define canine homologues of human CD antigens: summary of the first international canine leucocyte antigen workshop (CLAW). Tissue Antigens 43: 137-154.
  2. Brodersen, R. et al. (1998) Analysis of the immunological cross reactivities of 213 well characterized monoclonal antibodies with specificities against various CD markers of human and 11 animal species. Vet. Immunol. Immunopathol. 64: 1-13.
  3. Dean, G. A. et al. (1996) Proviral burden and infection kinetics of feline immunodeficiency virus in lymphocyte subsets of blood and lymph node. J. Virol. 70: 5165 - 5169.
  4. Faldyna, M. et al. (2004) Lymphocyte subsets in synovial fluid from clinically healthy joints of dogs. Acta Vet. Brno 73: 73-78.
  
• Gene Symbol: CR2
• Gene Full Name: Complement receptor type 2
• Alias Symbols: CR, C3DR, CD21, CVID7, SLEB9
• NCBI Gene Id: 1380
• Protein Name: Complement receptor type 2
• Description of Target: MOUSE ANTI CANINE CD21:RPE
  
• Uniprot ID: P20023
• Protein Accession #: NP_001006659.1; NP_001868.2
• Nucleotide Accession #: NM_001006658.2; NM_001877.4