CD335 antibody | VIV-KM1
Mouse anti Pig CD335 antibody, clone VIV-KM1 used for the identification of NK cells in influenza infected pigs by flow cytometry.
Image caption:
NK cell numbers in the blood of influenza infected pigs. Blood was taken from influenza A virus infected (n = 12) and control pigs (n = 9) on day 0−3 pi in a second experiment. (A) Isolated PBMCs were analysed by flow cytometry and live CD3− lymphocytes were gated as NKp46− or NKp46+ NK cells according to CD8α and NKp46 expression. Plots are taken from a representative control animal. Absolute numbers of (B) NKp46+ NK cells in PBMC were analysed by flow cytometry in control (left) and infected animals (right). (C) Results for the NKp46+ cells in the two groups were compared on each sampling day. (D) NKp46− NK cells in PBMC in control (left) and infected animals (right). (E) NKp46− NK cells were compared for the two groups. Each line in (B) and (D) represents one animal. **p≤0.01.
From: Forberg H, Hauge AG, Valheim M, Garcon F, Nunez A, et al. (2014)
Early Responses of Natural Killer Cells in Pigs Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus.
PLoS ONE 9(6): e100619.
Mouse anti Pig CD335 antibody, clone VIV-KM1 used for the identification of NK cells in influenza infected pigs by flow cytometry.
Image caption:
Percentages of NK cells and expression of NKp46 and CD25 in lung tissue. Mononuclear cells were isolated from lung tissue of pigs infected with influenza A virus (n = 12) and control animals (n = 6) during the first 3 days pi and analysed by flow cytometry. (A) Live CD3− lymphocytes were gated as described in Fig 2. NK cells were gated according to CD8α and NKp46 expression and defined as NKp46−, NKp46int or NKp46high cells. Plot shown is from a representative control animal. (B) Proportions of NKp46− (green), NKp46int (blue) and NKp46high (purple) NK cells in individual animals, shown as percentages of gated cells in lymphocytes. (C) Percentages of NKp46− (left), NKp46int (middle) and NKp46high (right) NK cells among lymphocytes. (D) Median fluorescence intensity (MFI) in the NKp46− gate (left), the NKp46+ gate (middle) and in the NKp46high gate (right) are shown. (E) CD25+ cells were gated in the NKp46− and NKp46int NK cells (left) and in the NKp46high NK cells (right). Plots shown are from a representative control animal. (F) The percentages of CD25+ cells in each gate were calculated in control animals (green), infected animals from day 1 (purple), day 2 (blue) and day 3 (pink) pi. *p≤0,05, **p≤0.01.
From: Forberg H, Hauge AG, Valheim M, Garcon F, Nunez A, et al. (2014) Early Responses of Natural Killer Cells in Pigs Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus.
PLoS ONE 9(6): e100619.
Mouse anti Pig CD335 antibody, clone VIV-KM1 used for the identification of NK cells in influenza infected pigs by immunofluorescence.
Image caption:
NKp46+ cells in the lungs of influenza virus infected pigs. Lung tissue sections from pigs infected with influenza A virus and control pigs were stained with immunofluorescence markers for cytokeratin (blue), NKp46 (green) and influenza A virus NP (red). NKp46+ cells were counted in areas were influenza A virus NP was (A) detected and (B) not detected. Representative pictures taken from the same animal on day 1 pi are shown. Arrows point at NKp46+ cells. Immunofluorescence staining, 200x. (C) Plot shows number of NKp46+ cells per 0,1 mm2 in sections (n = 24 per animal) from control animals (n = 6) and in areas with and without virus in infected animals (n = 4 per day) calculated as described in Material and Methods. Groups with different letters differ significantly (p≤0.05). (D) NKp46+ cells in the lumen of a bronchus (BL). Arrows point at the epithelial lining. Representative picture of luminal exudate, taken from an infected animal on day 2 pi. Insert shows NKp46+ and influenza A virus NP+ cell in the lung tissue of an infected animal on day 1 pi. Immunofluorescence staining, 400x.
From: Forberg H, Hauge AG, Valheim M, Garcon F, Nunez A, et al. (2014) Early Responses of Natural Killer Cells in Pigs Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus.
PLoS ONE 9(6): e100619.
Mouse anti Pig CD335 antibody, clone VIV-KM1 used for the identification of NK cells in influenza infected pigs by immunofluorescence.
Image caption:
Staining for apoptosis in the lungs. Lung tissue sections from animals infected with influenza A virus (n = 12) were stained with immunofluorescence markers against (A) NKp46(green), (B) influenza A NP(red) and (C) the apoptosis marker caspase-3(blue). (D) Overlay displaying simultaneously influenza A virus NP+ and caspase-3+ cells as purple (arrows). Representative of virus infected bronchiole at day 1 pi. Immunofluorescence staining, 400x.
From: Forberg H, Hauge AG, Valheim M, Garcon F, Nunez A, et al. (2014) Early Responses of Natural Killer Cells in Pigs Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus.
PLoS ONE 9(6): e100619.
Mouse anti Pig CD335 antibody, clone VIV-KM1 used for the identification of NK cells in influenza infected pigs by immunofluorescence.
Image caption:
Counting of NKp46+ cells. Numbers of NKp46+ cells per area were counted in lung tissue sections from control animals and influenza A virus infected animals as described in Material and Methods. Representative picture of area with virus from infected animal. Immunofluorescence staining, 200x.
From: Forberg H, Hauge AG, Valheim M, Garcon F, Nunez A, et al. (2014) Early Responses of Natural Killer Cells in Pigs Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus.
PLoS ONE 9(6): e100619.
Mouse anti Pig CD335 antibody, clone VIV-KM1 used for the evaluation of CD335 expression on porcine cells by flow cytometry.
Image caption:
Varying expression of NKp46, CD8α, CD16 and CD27 on NK-cell subsets in blood and spleen. (A) Following five-colour staining, PBMC and splenocytes were gated on CD3- cells and further subgated according to their NKp46/CD8α expression pattern in NKp46- and NKp46+ NK cells in blood (upper graph) and NKp46-, NKp46+ and NKp46high NK cells in spleen (lower graph). (B) 14 healthy 6–7 month old pigs were investigated for NKp46 and CD8α expression levels in the NKp46-defined NK-cell subsets. Box-plots show the mean fluorescence intensity of the two markers. (C) NK-cell subsets defined in (A) were further analysed for their expression of the surface markers CD16 and CD27. Histograms show the expression of the two markers within the respective NKp46-defined subsets (CD8α+NKp46-: blue histograms, CD8α+NKp46+: green histograms, CD8αdim/-NKp46high: red histograms) in blood (upper graphs) and spleen (lower graphs) according to the corresponding isotype control (grey histrograms with dotted lines). Box-plots show the mean fluorescence intensity of CD16 and CD27 of the NKp46-defined NK-cell subsets in blood and spleen of 14 healthy 6–7 month old pigs. (B + C) Significant differences between the subsets in blood or spleen are indicated (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).
From: Mair KH, Müllebner A, Essler SE, Duvigneau JC, Storset AK, Saalmüller A, Gerner W. Porcine CD8αdim/-NKp46high NK cells are in a highly activated state.
Vet Res. 2013 Mar 1;44:13.
Mouse anti Pig CD335 antibody, clone VIV-KM1 used for the evaluation of CD335 expression on porcine cells by flow cytometry.
Image caption:
Varying expression of NKp46, CD8α, CD16 and CD27 on NK-cell subsets in blood and spleen. (A) Following five-colour staining, PBMC and splenocytes were gated on CD3- cells and further subgated according to their NKp46/CD8α expression pattern in NKp46- and NKp46+ NK cells in blood (upper graph) and NKp46-, NKp46+ and NKp46high NK cells in spleen (lower graph). (B) 14 healthy 6–7 month old pigs were investigated for NKp46 and CD8α expression levels in the NKp46-defined NK-cell subsets. Box-plots show the mean fluorescence intensity of the two markers. (C) NK-cell subsets defined in (A) were further analysed for their expression of the surface markers CD16 and CD27. Histograms show the expression of the two markers within the respective NKp46-defined subsets (CD8α+NKp46-: blue histograms, CD8α+NKp46+: green histograms, CD8αdim/-NKp46high: red histograms) in blood (upper graphs) and spleen (lower graphs) according to the corresponding isotype control (grey histrograms with dotted lines). Box-plots show the mean fluorescence intensity of CD16 and CD27 of the NKp46-defined NK-cell subsets in blood and spleen of 14 healthy 6–7 month old pigs. (B + C) Significant differences between the subsets in blood or spleen are indicated (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).
From: Mair KH, Müllebner A, Essler SE, Duvigneau JC, Storset AK, Saalmüller A, Gerner W. Porcine CD8αdim/-NKp46high NK cells are in a highly activated state.
Vet Res. 2013 Mar 1;44:13.
Mouse anti Pig CD335 antibody, clone VIV-KM1 used for the evaluation of CD335 expression on porcine cells by flow cytometry.
Image caption:
Splenic NKp46high NK cells produced the highest levels of IFN-γ. (A + B) Intracellular cytokine staining for IFN-γ production in NKp46-defined NK cells within PBMC (upper graphs) and splenocytes (lower graphs) by four-colour FCM after 24 h in vitro stimulation with rhIL-2, rpIL-12 and rpIL-18 or medium. CD3- lymphocytes were gated (not shown) and NKp46/CD8α defined total NK cells were further subgated for IFN-γ production. IFN-γ producing NK cells were gated according to NKp46 expression levels (CD8α+NKp46-: blue, CD8α+NKp46+: green, CD8αdim/-NKp46high: red). (A) Numbers indicate the percentage of IFN-γ+ NK cells within respective gates. Results are representative for experiments with four different animals. (B) Percentage of IFN-γ+ cells (left graph) and mean fluorescence intensity of IFN-γ+ cells (right graph) within the different NK-cell subsets in blood and spleen of four animals are shown. (C) Supernatants of cytokine stimulated FACS-sorted CD3-CD8α+NKp46- and CD3-CD8α+NKp46+ NK cells from blood and CD3-CD8α+NKp46-, CD3-CD8α+NKp46+ and CD3-CD8αdim/-NKp46high NK cells from spleen were tested for IFN-γ production in ELISA following 24 h in vitro stimulation with rhIL-2, rpIL-12 and rpIL-18. Data on the left are from one representative animal and displayed as the mean of duplicates ± SD. IFN-γ production in experiments with four animals analysed is shown on the right. Mean values are represented by a black bar. (B + C) Significant differences between the subsets in blood or spleen are indicated (* = p < 0.05, ** = p < 0.01).
From: Mair KH, Müllebner A, Essler SE, Duvigneau JC, Storset AK, Saalmüller A, Gerner W. Porcine CD8αdim/-NKp46high NK cells are in a highly activated state.
Vet Res. 2013 Mar 1;44:13.
Mouse anti Pig CD335 antibody, clone VIV-KM1 used for the evaluation of CD335 expression on porcine cells by flow cytometry.
Image caption:
Analysis of degranulation capacity in NKp46-defined NK-cell subsets in blood. The cytolytic capacity of NKp46-defined NK-cell subsets (CD8α+NKp46-: blue, CD8α+NKp46+: green) isolated from blood was analysed after receptor triggering. Cells were stimulated with rhIL-2 and rpIL-15 overnight. Triggering of NK-receptors was performed by using monoclonal antibodies against NKp46, CD16 or a combination of both. Irrelevant isotype-matched antibody served as negative control. NK-cell receptor mediated degranulation was assessed by measuring the expression of CD107a on the cell surface by four-colour flow cytometry after one hour incubation. CD107a expression was measured on CD3- lymphocytes (not shown), followed by gating on the respective NKp46-defined NK subsets. (A) Numbers indicate the percentage of CD107a+ cells and the mean fluorescence intensity of CD107a within respective NKp46-gates. Results are representative of experiments with five different animals. (B) CD107a expression analyses of five animals analysed. The proportion of CD107a+ cells within the different NKp46-defined subsets is shown in the upper graphs. Percentage of CD107a+ NK cells was calculated by subtracting spontaneous degranulation observed in cultures stimulated with isotype-control antibodies from the frequency of CD107a+ cells in cultures stimulated with NKp46 and/or CD16 mAbs. The lower graphs show the mean fluorescence intensity of CD107a within the respective subsets. Mean values are represented by a black bar. Significant differences between the subsets are indicated (* = p < 0.05, ** = p < 0.01).
From: Mair KH, Müllebner A, Essler SE, Duvigneau JC, Storset AK, Saalmüller A, Gerner W. Porcine CD8αdim/-NKp46high NK cells are in a highly activated state.
Vet Res. 2013 Mar 1;44:13.
Mouse anti Pig CD335 antibody, clone VIV-KM1 used for the evaluation of CD335 expression on porcine cells by flow cytometry.
Image caption:
NKp46high NK cells in spleen showed the highest cytolytic capacity. The cytolytic capacity of NKp46-defined NK-cell subsets (CD8α+NKp46-: blue, CD8α+NKp46+: green, CD8αdim/-NKp46high: red) isolated from spleen was analysed after receptor-mediated degranulation. Cells were stimulated and gated for flow cytometric analysis as outlined in Figure 4. (A) Numbers indicate the percentage of CD107a+ cells and the mean fluorescence intensity of CD107a within respective NKp46-gates. Results are representative of experiments with five different animals. (B) CD107a expression analyses of five animals analysed. The proportion of CD107a+ cells within the different NKp46-defined subsets is shown in the upper graphs. Percentage of CD107a+ NK cells was calculated by subtracting spontaneous degranulation observed in cultures stimulated with isotype-control antibodies from the frequency of CD107a+ cells in cultures stimulated with NKp46 and/or CD16 mAbs. The lower graphs show the mean fluorescence intensity of CD107a within the respective subsets. Mean values are represented by a black bar. Significant differences between the subsets are indicated (* = p < 0.05, ** = p < 0.01).
From: Mair KH, Müllebner A, Essler SE, Duvigneau JC, Storset AK, Saalmüller A, Gerner W. Porcine CD8αdim/-NKp46high NK cells are in a highly activated state.
Vet Res. 2013 Mar 1;44:13.
Mouse anti Pig CD335 antibody, clone VIV-KM1 recognizes the porcine homologue of human CD335, also known as NKp46 and natural cytotoxicity triggering receptor 1 (NCR1), a member of the natural cytotoxicity receptor (NCR) family.
CD335 is a type I transmembrane protein, with two extracellular C2-type immunoglobulin-like domains, which functions as an activating receptor and is involved in the control of viral infection and tumor development.
Until recently little has been known about porcine and veterinary NK cells. CD335 is expressed by human natural killer cells (Sivori, S. et al.1997) and the development of monoclonal antibodies to bovine CD335 (clone ASK1) (Storset et al. 2004) and ovine CD335 (clone EC1.1) (Connelley et al.2011) have enabled researchers to identify and better understand ruminant NK cells. Clone VIV-KM1 is the first monoclonal developed to specifically identify porcine CD335 and provides a reagent to facilitate a better understanding of the pig immune system and aid in the understanding of the role of NK cells in host pathogen defence. Studies using VIV-KM1 have shown that, within the pig, CD335 is not universally expressed by all NK cells and that expression of this marker on NK cells may be influenced by cytokine production (Mair et al. 2012).
In addition to clone VIV-KM1, clones AKS1, which recognizes CD335 (NKp46) in bovine and other ruminants, and EC1.1, which recognizes ovine and caprine CD335, are also available from Bio-Rad.
CD335 is a type I transmembrane protein, with two extracellular C2-type immunoglobulin-like domains, which functions as an activating receptor and is involved in the control of viral infection and tumor development.
Until recently little has been known about porcine and veterinary NK cells. CD335 is expressed by human natural killer cells (Sivori, S. et al.1997) and the development of monoclonal antibodies to bovine CD335 (clone ASK1) (Storset et al. 2004) and ovine CD335 (clone EC1.1) (Connelley et al.2011) have enabled researchers to identify and better understand ruminant NK cells. Clone VIV-KM1 is the first monoclonal developed to specifically identify porcine CD335 and provides a reagent to facilitate a better understanding of the pig immune system and aid in the understanding of the role of NK cells in host pathogen defence. Studies using VIV-KM1 have shown that, within the pig, CD335 is not universally expressed by all NK cells and that expression of this marker on NK cells may be influenced by cytokine production (Mair et al. 2012).
In addition to clone VIV-KM1, clones AKS1, which recognizes CD335 (NKp46) in bovine and other ruminants, and EC1.1, which recognizes ovine and caprine CD335, are also available from Bio-Rad.
Product Details
- Target Species
- Pig
- Product Form
- Purified IgG conjugated to Alexa Fluor® 488 - liquid
- Product Form
- Purified IgG conjugated to Allophycocyanin (APC) - lyophilised
- Product Form
- Purified IgG - liquid
- Reconstitution
- Reconstitute with 1.0 ml distilled water
- Preparation
- Purified IgG prepared by affinity chromatography on Protein A from tissue culture supernatant
- Preparation
- Purified IgG prepared by affinity chromatography on Protein A from tissue culture supernatant
- Preparation
- Purified IgG prepared by affinity chromatography on Protein A from tissue culture supernatant
- Buffer Solution
- Phosphate buffered saline
- Buffer Solution
- Phosphate buffered saline
- Buffer Solution
- Phosphate buffered saline
- Preservative Stabilisers
- 0.09% Sodium Azide (NaN3)
1% Bovine Serum Albumin - Preservative Stabilisers
- 0.09% Sodium Azide (NaN3)
1% Bovine Serum Albumin
5% Sucrose - Preservative Stabilisers
- 0.09% Sodium Azide (NaN3)
- Carrier Free
- Yes
- Immunogen
- Fusion protein consisting of the extracellular region of porcine CD335
- Approx. Protein Concentrations
- IgG concentration 0.05 mg/ml
- Approx. Protein Concentrations
- IgG concentration 1.0 mg/ml
- Fusion Partners
- Spleen cells from immunised Balb/c mice were fused with cells of the SP2/0 myeloma cell line
Storage Information
- Storage
- Store at +4oC or at -20oC if preferred.
This product should be stored undiluted.
Storage in frost-free freezers is not recommended. This product is photosensitive and should be protected from light.
Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use. - Storage
- Prior to reconstitution store at +4oC.
After reconstitution store at +4oC.
DO NOT FREEZE.
This product should be stored undiluted. This product is photosensitive and should be protected from light. Should this product contain a precipitate we recommend microcentrifugation before use. - Storage
- Store at +4oC or at -20oC if preferred.
This product should be stored undiluted.
Storage in frost-free freezers is not recommended. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use - Shelf Life
- 18 months from date of despatch
- Shelf Life
- 12 months from date of reconstitution
- Shelf Life
- 18 months from date of despatch.
More Information
- Acknowledgements
- This product is provided under an intellectual property licence from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchase product solely in research, excluding contract research or any fee for service research, and the buyer must not sell or otherwise transfer this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; (c) manufacturing or quality assurance or quality control, or (d) resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad CA 92008 USA or outlicensing@thermofisher.com
- Regulatory
- For research purposes only
Applications of CD335 antibody
| Application Name | Verified | Min Dilution | Max Dilution |
|---|---|---|---|
| Flow Cytometry | Neat | 1/10 | |
| Flow Cytometry | Neat | ||
| Flow Cytometry | 1/50 | 1/200 | |
| Immunofluorescence |
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
Where this antibody has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. It is recommended that the user titrates the antibody for use in their own system to a concentration equivalent to their test reagent.
- Flow Cytometry
- Use 10ul of the suggested working dilution to label 106 cells in 100ul
- Flow Cytometry
- Use 10ul of the suggested working dilution to label 106 cells in 100ul
- Flow Cytometry
- Use 10ul of the suggested working dilution to label 106 cells in 100ul.
Copyright © 2018 Bio-Rad Antibodies (formerly AbD Serotec)
Secondary Antibodies Available
Negative Isotype Controls Available
| Description | Product Code | Pack Size | Applications | List Price | Quantity |
|---|---|---|---|---|---|
| Mouse IgG1 Negative Control:Alexa Fluor® 488 | MCA928A488 | 100 Tests/1ml | F | ||
| Mouse IgG1 Negative Control:APC | MCA928APC | 100 Tests | F | ||
| Mouse IgG1 Negative Control | MCA928 | 100 Tests | F |
Useful Reagents Available
| Description | Product Code | Pack Size | Applications | List Price | Quantity |
|---|---|---|---|---|---|
| Mouse anti Pig CD16:RPE | MCA1971PE | 100 Tests | F | ||
| Mouse anti Bovine CD335 | MCA2365GA | 0.1 mg | F IF WB | ||
| Mouse anti Pig CD3:RPE | MCA5951PE | 100 Tests | F | ||
| Mouse anti Pig CD27:RPE | MCA5973PE | 100 Tests | F | ||
| Human anti Pig Interferon Gamma | HCA090 | 0.1 mg | E | ||
| Mouse anti Pig CD45:FITC | MCA1222F | 100 Tests | F | ||
| Mouse anti Pig wCD8 Alpha:RPE | MCA1223PE | 100 Tests | F | ||
| Rat anti Human CD3:Alexa Fluor®647 | MCA1477A647 | 100 Tests/1ml | F* | ||
| Rat anti Human CD3:Pacific Blue® | MCA1477PB | 100 Tests/1ml | F* | ||
| Mouse anti Pig CD25 | MCA1736GA | 0.1 mg | C F | ||
| Mouse anti Pig CD107a:Alexa Fluor® 647 | MCA2315A647 | 100 Tests/1ml | F* | ||
| Mouse anti Pig CD107a:FITC | MCA2315F | 0.1 mg | F* | ||
| Mouse anti Pig CD3:FITC | MCA5951F | 0.1 mg | F | ||
| Mouse anti Pig CD3:RPE | MCA5951PE | 100 Tests | F |
Product Specific References
References for CD335 antibody
-
Forberg, H. et al. (2014) Early responses of natural killer cells in pigs experimentally infected with 2009 pandemic H1N1 influenza A virus.
PLoS One. 9 (6): e100619. -
Mair, K.H. et al. (2013) Porcine CD8αdim/-NKp46high NK cells are in a highly activated state.
Vet Res. 44: 13. -
Mair, K.H. et al. (2012) NKp46 expression discriminates porcine NK cells with different functional properties.
Eur J Immunol. 42 (5): 1261-71.
Further Reading
-
Connelley, T. et al. (2011) NKp46 defines ovine cells that have characteristics corresponding to NK cells.
Vet Res. 42: 37. -
Storset, A.K. et al. (2004) NKp46 defines a subset of bovine leukocytes with natural killer cell characteristics.
Eur J Immunol. 34 (3): 669-76. -
Sivori, S. et al. (1997) p46, a novel natural killer cell-specific surface molecule that mediates cell activation.
J Exp Med. 186 (7): 1129-36.
