hello this is Jasmine Trigg and I'm a scientist  at Sartorius today I'll be discussing a poster  
hello this is Jasmine Trigg and I'm a scientist  at Sartorius today I'll be discussing a poster  

previously presented at the American Association  of meteorologists conference and the post was  
previously presented at the American Association  of meteorologists conference and the post was  

titled high throughput quantification of antibody  dependent phagocytosis use in lifestyle analysis  
titled high throughput quantification of antibody  dependent phagocytosis use in lifestyle analysis  

phagocytosis a specific form of endocytosis  is a critical component of both innate and  
phagocytosis a specific form of endocytosis  is a critical component of both innate and  

adaptive immune responses the uptake and  clear clearance of viable tumor cells can  
adaptive immune responses the uptake and  clear clearance of viable tumor cells can  

be promoted with monoclonal antibodies via  antibody dependent cellular phag cytosis also  
be promoted with monoclonal antibodies via  antibody dependent cellular phag cytosis also  

known as adcp all through the blockade  of don't eat new signals such as cd47  
known as adcp all through the blockade  of don't eat new signals such as cd47  

these mechanisms hold immuno therapeutic promise  and are studied extensively in drug development  
these mechanisms hold immuno therapeutic promise  and are studied extensively in drug development  

so here we have developed and validated  an in vitro assay for the high throughput  
so here we have developed and validated  an in vitro assay for the high throughput  

quantification of Phagocytosis using  the ink site lifestyle analysis system  
quantification of Phagocytosis using  the ink site lifestyle analysis system  

the ink sitebag cytosis assay combines Frodo  for ink site reagents and integrated image-based  
the ink sitebag cytosis assay combines Frodo  for ink site reagents and integrated image-based  

fluorescence measurements in a simple mix  and read protocol the data that I'll be  
fluorescence measurements in a simple mix  and read protocol the data that I'll be  

showing today exemplify that liveso analysis is  a powerful tool for quantitative morphological  
showing today exemplify that liveso analysis is  a powerful tool for quantitative morphological  

and functional assessment of adcp and this is  amenable to screening for therapeutic candidates  
and functional assessment of adcp and this is  amenable to screening for therapeutic candidates  

the introsite lifestyle analysis system is a  fully automated phase contrast and multi-color  
the introsite lifestyle analysis system is a  fully automated phase contrast and multi-color  

fluorescent system that resides inside a standard  cell culture incubator for optimal cell viability  
fluorescent system that resides inside a standard  cell culture incubator for optimal cell viability  

the system is designed to scan plates and  flasks repeatedly over time in combination  
the system is designed to scan plates and  flasks repeatedly over time in combination  

with our software this offers fast flexible and  Powerful control for continuous lifestyle analysis  
with our software this offers fast flexible and  Powerful control for continuous lifestyle analysis  

and this comprises image acquisition processing  and data visualization we also offer Suite of  
and this comprises image acquisition processing  and data visualization we also offer Suite of  

reagents and consumables that are non-perturbing  and these are cell labeling and Reporting reagents
reagents and consumables that are non-perturbing  and these are cell labeling and Reporting reagents

so phagocytosis is a process by which certain  living cells are able to engulf and digest other  
so phagocytosis is a process by which certain  living cells are able to engulf and digest other  

cells or particles Frodo reagents for incusite  phagocytosis use aph sensitive conjugated  
cells or particles Frodo reagents for incusite  phagocytosis use aph sensitive conjugated  

fluorophore that has been Incorporated to enable  lifestyle Imaging of phagocytic events the assay  
fluorophore that has been Incorporated to enable  lifestyle Imaging of phagocytic events the assay  

principle shown here is based on a low level of  presence of frodo-labeled material in media that's  
principle shown here is based on a low level of  presence of frodo-labeled material in media that's  

around pH 7.4 which following engulfment and  figure lysosome formation results in the increase  
around pH 7.4 which following engulfment and  figure lysosome formation results in the increase  

in the acidic environment and this leads to an  increase in the fluorescence intensity within  
in the acidic environment and this leads to an  increase in the fluorescence intensity within  

cytoplasm of the cell using the ink site lives  analysis system this increase in fluorescence  
cytoplasm of the cell using the ink site lives  analysis system this increase in fluorescence  

can be automatically Quantified over time with  integrated software to segment the areas of high  
can be automatically Quantified over time with  integrated software to segment the areas of high  

intensity additionally they observed phagocytic  signals can be confirmed using the ink site HD  
intensity additionally they observed phagocytic  signals can be confirmed using the ink site HD  

phase contrast images and these allow for direct  visualization of cell engulfment in real time  
phase contrast images and these allow for direct  visualization of cell engulfment in real time  

for example in the figure in the right here  photo labeled apoptotic gercat cells were  
for example in the figure in the right here  photo labeled apoptotic gercat cells were  

co-cultured with j7748.1 mouse macrophages  at a range of Target to effector ratios  
co-cultured with j7748.1 mouse macrophages  at a range of Target to effector ratios  

phase and fluorescent images that 20x were  acquired using the exercise system and the  
phase and fluorescent images that 20x were  acquired using the exercise system and the  

phagocytosis was Quantified using the  fluorescent segmentation mask which is  
phagocytosis was Quantified using the  fluorescent segmentation mask which is  

shown in the images here in blue when we look  at the time course we can see that there's a  
shown in the images here in blue when we look  at the time course we can see that there's a  

density dependent increase in red fluorescence  following engulfing over the 24-hour time period
density dependent increase in red fluorescence  following engulfing over the 24-hour time period

tumor cells can be targeted with  prophetic monoclonal antibodies which  
tumor cells can be targeted with  prophetic monoclonal antibodies which  

engage host immune cells and promote  engulfment and clearance on the body  
engage host immune cells and promote  engulfment and clearance on the body  

one strategy for this is the blockage of don't eat  new signals such as cd47 and these are present on  
one strategy for this is the blockage of don't eat  new signals such as cd47 and these are present on  

the surface of tumor cells and enable evasion of  phagocytosis to investigate these don't eat new  
the surface of tumor cells and enable evasion of  phagocytosis to investigate these don't eat new  

signals frodo-labeled ccrfcm tumor cells were  treated with monoclonal antibodies targeting  
signals frodo-labeled ccrfcm tumor cells were  treated with monoclonal antibodies targeting  

cd47 or IGG ice type control and they were  co-cultured with bone marrow drive to macrophages  
cd47 or IGG ice type control and they were  co-cultured with bone marrow drive to macrophages  

if we look at the images here on the left  these are phase and fluorescence images  
if we look at the images here on the left  these are phase and fluorescence images  

for five microgram per ML anti-cd47 and these are  acquired using the ink site lives analysis system  
for five microgram per ML anti-cd47 and these are  acquired using the ink site lives analysis system  

the images show an increase in fluorescence as  these cells were engulfed a kinetic increase in  
the images show an increase in fluorescence as  these cells were engulfed a kinetic increase in  

frescence was observed with monoclonal antibody  mediated response being concentration dependent  
frescence was observed with monoclonal antibody  mediated response being concentration dependent  

to eliminate the possibility of anti-seed  47 treatment resulting in Target cell  
to eliminate the possibility of anti-seed  47 treatment resulting in Target cell  

apoptosis and inducing ephocytosis we also  performed phagocytosis and cell health assays  
apoptosis and inducing ephocytosis we also  performed phagocytosis and cell health assays  

here frodo-labeled ccrfcm cells were treated  with antecedented seven or campothesium which  
here frodo-labeled ccrfcm cells were treated  with antecedented seven or campothesium which  

acted as our apoptotic control and  co-cultured with j7748.1 mouse Mac  
acted as our apoptotic control and  co-cultured with j7748.1 mouse Mac  

phages we did this in the presence of the inksi  and xm5 dye and this is a cell Health reagent  
phages we did this in the presence of the inksi  and xm5 dye and this is a cell Health reagent  

we found that anti-city 47 treatment induced  phagocytosis without inciting apoptosis
we found that anti-city 47 treatment induced  phagocytosis without inciting apoptosis

to investigate phagocytosis of viable tumor  cells via antibody dependent cellulocytosis  
to investigate phagocytosis of viable tumor  cells via antibody dependent cellulocytosis  

we assessed two clinical monoclonal antibodies  targeted against cd20 these were rituximal and  
we assessed two clinical monoclonal antibodies  targeted against cd20 these were rituximal and  

subtraxima which is a trucks made by similar and  these are both the true approved treatments for  
subtraxima which is a trucks made by similar and  these are both the true approved treatments for  

cd20 positive B cell cancers photo labeled Ramos  target cells at a rate of densities from 100 to  
cd20 positive B cell cancers photo labeled Ramos  target cells at a rate of densities from 100 to  

12.5 K per well we're treated with anti-cv20  Moroccan antibodies for toximab or troxima  
12.5 K per well we're treated with anti-cv20  Moroccan antibodies for toximab or troxima  

or an igg-1 ice type control and then they were  co-cultured with bone marrow derived macrophages  
or an igg-1 ice type control and then they were  co-cultured with bone marrow derived macrophages  

if we look at the time course on the  left here this shows that traxma-induced  
if we look at the time course on the  left here this shows that traxma-induced  

um adcp response is both time and target cell  density dependent the results in the middle  
um adcp response is both time and target cell  density dependent the results in the middle  

show an increase in fluorescence for truxma and  rituximab compared to IGG control at all of the  
show an increase in fluorescence for truxma and  rituximab compared to IGG control at all of the  

target cell densities that we've studied  lastly we talk some AB treatment showed a  
target cell densities that we've studied  lastly we talk some AB treatment showed a  

concentration dependent increase in adcp using  the target cell density of 50k cells per wow
concentration dependent increase in adcp using  the target cell density of 50k cells per wow

we also assessed the effects of  anti-cd-20 monoclonal antibodies in  
we also assessed the effects of  anti-cd-20 monoclonal antibodies in  

inducing phagocytosis using primary  Mac phages as the affect the cells  
inducing phagocytosis using primary  Mac phages as the affect the cells  

monocytes isolated from Whole Bloods were  differentiated into macrophages using cytokines  
monocytes isolated from Whole Bloods were  differentiated into macrophages using cytokines  

frodo-labeled Ramos cells were treated with  monoclonal antibodies targeting anti-cd-20 again  
frodo-labeled Ramos cells were treated with  monoclonal antibodies targeting anti-cd-20 again  

here we use rituximab or truxma or IGG ice type  control and co-cultured these with the primary Mac  
here we use rituximab or truxma or IGG ice type  control and co-cultured these with the primary Mac  

phages that we've differentiated so the macrophage  differentiation itself was monitored and  
phages that we've differentiated so the macrophage  differentiation itself was monitored and  

Quantified using the integrated software of the  ink site Advanced Label 3 classification analysis  
Quantified using the integrated software of the  ink site Advanced Label 3 classification analysis  

and we can see the images of the classification  masks here so on the left we have images for the  
and we can see the images of the classification  masks here so on the left we have images for the  

classification mask for monocytes which was on  Day Zero with a pink outline and the right hand  
classification mask for monocytes which was on  Day Zero with a pink outline and the right hand  

image shows macrophages with a blue classification  um mask outline on day seven looking at the time  
image shows macrophages with a blue classification  um mask outline on day seven looking at the time  

course kinetic data this shows that rituximab  and troxima induced adcp in a concentration  
course kinetic data this shows that rituximab  and troxima induced adcp in a concentration  

dependent manner with similar kinetic profiles  being observed over the four hour time period  
dependent manner with similar kinetic profiles  being observed over the four hour time period  

foreign next we investigated some native  and engineered products map FC mutants  
foreign next we investigated some native  and engineered products map FC mutants  

frodo-labeled vammal cells were treated with  Native and engineered human with toxinant  
frodo-labeled vammal cells were treated with  Native and engineered human with toxinant  

Isotopes and to see the 20 igg-1 and cd20 igg-1  foots which is a non-fuco selected FC mutant and  
Isotopes and to see the 20 igg-1 and cd20 igg-1  foots which is a non-fuco selected FC mutant and  

anti-c20i21 and Q which is a non-glycosylated fc  mutant and they were co-cultured with bone marrow  
anti-c20i21 and Q which is a non-glycosylated fc  mutant and they were co-cultured with bone marrow  

drive Mac fetus with a 1 to 2.5 k effects Target  ratio if we look at the microplate graph on the  
drive Mac fetus with a 1 to 2.5 k effects Target  ratio if we look at the microplate graph on the  

left here this shows the kinetic response  for total red object area over four hours  
left here this shows the kinetic response  for total red object area over four hours  

we observed concentration dependent adcp response  for and cd20 igd1 monoclonal antibody and the FC  
we observed concentration dependent adcp response  for and cd20 igd1 monoclonal antibody and the FC  

mutated igg-1 foot but not for the igg-1 and Q  or IGG control and the ec50 values for these were  
mutated igg-1 foot but not for the igg-1 and Q  or IGG control and the ec50 values for these were  

shown in the table here these results align  with previously observed effector functions
shown in the table here these results align  with previously observed effector functions

additionally we've studied adcp using  adherent target cells that had varying  
additionally we've studied adcp using  adherent target cells that had varying  

her2 antigen expression profiles so adcp  was examined using her2 positive ae565 or  
her2 antigen expression profiles so adcp  was examined using her2 positive ae565 or  

her2 low MTA adherent target cells cells  were labeled with Frodo and then treated  
her2 low MTA adherent target cells cells  were labeled with Frodo and then treated  

with an anti-her2 monoclonal antibody and  co-cultured with bone Margaret macrophages  
with an anti-her2 monoclonal antibody and  co-cultured with bone Margaret macrophages  

if we look at the images Auntie her to induced  an increase in fluorescence for au565 cells but  
if we look at the images Auntie her to induced  an increase in fluorescence for au565 cells but  

little to no fluorescence was up there for MDA  mb23 cells the time course shows an adcp response  
little to no fluorescence was up there for MDA  mb23 cells the time course shows an adcp response  

for au565 cells and reveals that this occurs  in a time and concentration dependent manner  
for au565 cells and reveals that this occurs  in a time and concentration dependent manner  

and to her to induce adcp for a565 cells but  not for the MDA cells and this is consistent  
and to her to induce adcp for a565 cells but  not for the MDA cells and this is consistent  

with established correlations between  her to expression and adcp response
with established correlations between  her to expression and adcp response

so to conclude the photo reagents for ink site  in combination with the site lifestyle analysis  
so to conclude the photo reagents for ink site  in combination with the site lifestyle analysis  

system provides an image-based fluorescent  solution for visualization and quantification of  
system provides an image-based fluorescent  solution for visualization and quantification of  

thyocytosis our experimental findings demonstrate  antibody mediated cell life cytosis across a  
thyocytosis our experimental findings demonstrate  antibody mediated cell life cytosis across a  

range of effector Target cell types and they  substantiate the recognized prophagocytic effects  
range of effector Target cell types and they  substantiate the recognized prophagocytic effects  

of monoclonal antibodies such as anti-cb47  anticed 20 and also anti-air2 taken together  
of monoclonal antibodies such as anti-cb47  anticed 20 and also anti-air2 taken together  

these data exemplify that lifestyle analysis is a  powerful tool for quantitative morphological and  
these data exemplify that lifestyle analysis is a  powerful tool for quantitative morphological and  

functional assessment of adcp which is amenable  to screening for therapeutic candidates thank you
functional assessment of adcp which is amenable  to screening for therapeutic candidates thank you