Dual staining of pig peripheral blood lymphocytes with Mouse anti Pig CD335:Alexa Fluor488 and Mouse anti Pig wCD8a:RPE

Published investigator image: 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, CD8a, 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/CD8a 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 CD8a 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 (CD8a+NKp46-: blue histograms, CD8a+NKp46+: green histograms, CD8adim/-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, Mllebner A, Essler SE, Duvigneau JC, Storset AK, Saalmller A, Gerner W. Porcine CD8adim/-NKp46high NK cells are in a highly activated state. Vet Res. 2013 Mar 1;44:13.

Published investigator image: 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-?. (A + B) Intracellular cytokine staining for IFN-? 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/CD8a defined total NK cells were further subgated for IFN-? production. IFN-? producing NK cells were gated according to NKp46 expression levels (CD8a+NKp46-: blue, CD8a+NKp46+: green, CD8adim/-NKp46high: red). (A) Numbers indicate the percentage of IFN-?+ NK cells within respective gates. Results are representative for experiments with four different animals. (B) Percentage of IFN-?+ cells (left graph) and mean fluorescence intensity of IFN-?+ 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-CD8a+NKp46- and CD3-CD8a+NKp46+ NK cells from blood and CD3-CD8a+NKp46-, CD3-CD8a+NKp46+ and CD3-CD8adim/-NKp46high NK cells from spleen were tested for IFN-? 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-? 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, Mllebner A, Essler SE, Duvigneau JC, Storset AK, Saalmller A, Gerner W. Porcine CD8adim/-NKp46high NK cells are in a highly activated state. Vet Res. 2013 Mar 1;44:13.

Published investigator image: 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.

Published investigator image: 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 CD8a 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.

Published investigator image: 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.

Published investigator image: 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.

Published investigator image: 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 CD8a 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.

Dual staining of pig peripheral blood lymphocytes with Mouse anti Pig CD335 detected with Goat anti Mouse IgG (H/L):FITC (STAR117F), and Mouse anti Pig wCD8a:RPE