1. Cytotoxicity in Mouse Macrophage RAW264.7 cells against Mouse Mastocytoma P815 cells,
Mediated by IFN-alpha, LPS, or Both
Anastasia Belyakova
February 24th, 2016
Abstract

2. The immunological response of macrophages upon pathogenic attack is important in the
survival of an organism. The purpose of this experiment series was to analyze and quantify the
cytotoxic activity of the mouse macrophage cell line (RAW264.7) upon exposure to mouse
mastocytoma tumor cells (P815) as a model of the innate immune response mechanism by measuring
LDH release by lysed cells. To elucidate the effectiveness of the macrophage reaction, we diluted a
concentrated RAW cell solution into five samples, and prepared a 96 well plate to which we added a
constant amount of P815 cells, as well as mediators IFN-gamma, LPS, or both in order to determine
what combination would yield the best LDH release. Once the absorbances were run, cytotoxicity
could be calculated. We found that the highest percentage of cytotoxicity was for the 5:1 (RAW #2)
dilution at 13.9%, and the most effective cytotoxicity in the 3:1 RAW #5 mediator-affected killing
activity was for the co-culture with both LPS and IFN-gamma present in solution at 4.29%.
Introduction
The immune system, though complex, has a straightforward set of functions: to recognize
foreign infectious particles and invoke a systemic response to eradicate them. In order to survive, an
individual must have functional innate and adaptive immune response mechanisms if the body's
defense barriers are unsuccessful at preventing pathogenic entry. The innate response is termed “natural
resistance” because it is fast and non-specific – as long as the effector cells recognize invading cells as
non-self (Schwander, 6). Macrophages are the cells that are involved in antigen recognition and
cytokine signaling to start the immune response cycle once the foreign particles breach barriers and
invade host tissue.
Once the macrophage comes into contact with a foreign particle, it performs a pattern
recognition. The surface of many pathogenic microorganisms have a conserved pattern of molecular
structures that are not present on the host's cells, and macrophages have pattern recognition receptors
(PRR) that are used to recognize the pathogen-associated molecular patterns (PAMPS) in order to then

3. enact the proper systemic response of secreting cytokines and chemokines (Janeway, 11-12). Then
once the inflammatory response has been induced, the macrophages, along with other inflammatory
cells, engulf and lyse the target foreign cells, thereby eradicating the infection. In order to test the
effectiveness of the macrophages against invading cells, a CytoTox 96 Non-Radioactive Cytotoxicity
Assay can be performed. This method colorimetrically measures lactate dehydrogenase (LDH) released
by lysed target cells, with which we can quantitatively measure the cytotoxic activity of the
macrophages (Schwander, 18). Cytotoxic activity is not only induced by the presence of pathogens, but
can be mediated as well. In our experiment, we looked the effect of LPS and IFN-gamma, and
compared their effect on the macrophage activity by themselves and in combination. Our hypothesis
was that the highest effector:target cell ratio would have the most effective cytotoxic activity, and that
the combination of LPS and IFN-gamma with the RAW #5 3:1 ratio would also have the highest killing
activity as opposed to lacking mediators or having only one in solution.
Materials and Methods
In the series of experiments performed to understand the role of macrophages, it was important
to observe their phagocytic ability. Upon extracting macrophages and lymphocytes (peritoneal exudate
cells, PEC) out of the peritoneum of a mouse and exposing them to latex pellets, we prepared Petri
dishes and looked at the macrophage reaction to the foreign particles. As expected, the macrophages
adhered to the pellets. Then in the second experiment of the macrophage series, we prepared the
macrophage dilutions. We used a cell culture medium composed of 5ml DMEM/F12 (without
indicator) and 3% fetal calf serum in a 15ml conical tube. The stock concentration of RAW264.7 cells
was [2.04 x 106 cells/ml], which we diluted into five eppendorf tubes (#1-5) with tube RAW #1 as the
starting macrophage concentration, tube RAW #2 was diluted to [2.4 x 105 cells/ml], tubes RAW #3
and RAW #5 diluted to [1.03 x 106 cells/ml] and tube RAW #4 diluted to [2.67 x 104 cells/ml]. Once
the dilutions were prepared and mixed, 100 ul of each dilution were added to their respective wells on

4. the 96 microtiter well plate, and the mediators LPS and IFN-gamma were added to respective wells
before incubating for an hour at 37 degrees Celsius. After incubation, 100 ul of P815 target cells were
added to the wells. The ratio of effector to target cells for the five concentrations were 10:1, 5:1, 3:1,
1:1 and 3:1 for RAW #1-5, respectively. The well plate was again incubated for approximately sixteen
hours before the addition of 20ul 10X lysis solution to determine the max lyses, and the plates were
centrifuged at 1200 rpm for five minutes, in order to release the LDH into the supernatant (and leave
irrelevent particles coagulated at the bottom. After this, 50ul of supernatant solutions were added to a
new well plate and frozen at -20 Celsius to hinder the progress of a reaction.
In the final experiment, the well plate was prepared for analysis of macrophage killing. First
50ul of the positive control of LDH at 1:10,000 dilution was added to the A1 well to use as reference
for the rest of the plate. Then 50ul of substrate mix was added to all the wells in the assay, and
incubated in a dark, room temperature environment for half an hour. To end the lysis reaction, 50ul of
stop solution was then added to all the wells and absorbance of the well plate was measured and
recorded at an absorbance of 490nm. Then the absorbance values were recorded and cytotoxicity
results were calculated and compared.
Results
Table 1: Absorbance values of 96 microtiter plate quantitative measurement of LDH release
1 3 4 5 6 8 9 10 11
A 0.7982 0.5678 1.5949 0.6036
B 1.4523 1.21
C 0.1052 0.1161
D 0.2148 0.1915
E 0.3862 0.2599 0.2073 0.1446 0.2779 0.213 0.2069 0.2579

5. F 0.47 0.3438 0.2932 0.2277 0.3146 1.1888 0.1406 0.1942
G 0.5785 0.6747 0.3838 0.2451 0.3257 0.3207 0.4564 0.4803
Table 1 shows the raw data of absorbance values measured by the Cytotox 96 Non-Radioactive
Cytotoxicity Assay, measured at 490nm. Well A1 is the positive control LDH reference to which all
other LDH release absorbances are compared.
Table 2: Percent Cytotoxicity Values for RAW #1-5 and mediated RAW #5
RAW Effector:Target % Cytotoxicity
#1 10:1 4.04
#2 5:1 13.9
#3 3:1 3.43
#4 1:1 -0.06
#5 (no mediator) 3:1 0.47
#5 LPS(+) 3:1 -9.88
#5 IFN-gamma(+) 3:1 2.18
#5 LPS(+) IFN-gamma(+) 3:1 4.29
Table 2 shows the comparison of cytotoxicity among the RAW264.7 dilutions #1-4 and variably
mediated RAW #5 wells. The highest cytotoxicity values were for RAW #2 at 13.9%, and at RAW #5
LPS(+) and IFN-gamma(+).
Discussion
Understanding how the immune system functions correctly is imperative to dealing with a
compromised immune system. In this series of experiments, we tested and analyzed the cytotoxic
effects of macrophages upon exposure to pathogenic cells, and compared cytotoxic effectiveness of the
effector cells in different ratios to target cells, as well as under mediation. What was interesting to
observe was the highest (10:1) effector to target cell ratio did not correspond to the prediction that the

6. cytotoxicity percentage would be highest. In fact the 5:1 ratio produced the best result. The reasoning
for this may be because the target cells were so much outnumbered, they were lysed, and the effector
cells for the most part were not – whereas in the other ratios where target cells were higher in ratio,
there was more of an LDH release and therefore a change in cytotoxic effect. In the 5:1 ratio, the
%cytotocity was at 13.9%, as compared to the 4.04% with the 10:1 ratio. However, with a lower ratio,
there is clearly more of a strain on the RAW264.7 cells because there are more P815 cells to attack, and
inevitably that may cause more effector cell death as well. This is evident in the 1:1 ratio, where the
cytotoxicity is in fact -0.06%, showing that the effector cells struggled to fight the infection.
Alternatively, the mediated RAW #5 cell solutions behaved according to original predictions –
the co-culture combination of both LPS and IFN-gamma mediators produced the highest cytotoxic
percentage result at 4.29%. Surprisingly, the lowest cytotoxic effect was for the LPS(+) solution, which
resulted in a -9.88% cytotoxic effect – although LPS stimulates macrophage activity, the lack of IFN-
gamma slowed down the positive feedback of activating the macrophages fast enough.
The understanding of beneficial effector to target cell ratios and mediation helps in explaining
why the immune response is so important. As we can see, the earlier the immune cells recognize a
pathogen and respond to it, the less cytotoxic effect there is and the faster the infection is eradicated.
Due to the effectiveness of the macrophages in their role of recognition and inflammatory stimulation,
organisms are given a chance to overcome infections and survive.
References:
Covey, L., Firestein, B., Grudzien, E., Schwander, M., Xie, P., Immunology Laboratory Manual.
Rutgers University, Department of Cell Biology & Neuroscience 2016:6-18pp.
Murphy KM, P Travers, M Walport (Eds.) (2010) Janeway's Immunobiology. 8th Edition. New
York:Taylor & Francis, Inc. 11-12pp.