LPS Stimulation of THP-1 Cells and Measurement of TNF-α by ELISA

MIDHUN MADHU
B01660577
MSC BIOTECHNOLOGY

1. Introduction

THP-1 cells are a type of human monocytic cell line commonly used in biomedical
research, particularly in studies related to immunology, inflammation, and cancer
(Bosshart and Heinzelmann, 2016). These cells were derived from the peripheral
blood of a patient with acute monocytic leukemia. THP-1 cells exhibit properties
characteristic of monocytes, including adherence to plastic surfaces and the ability to
differentiate into macrophage-like cells in response to various stimuli. THP-1 cells
are widely used as a model system to study the molecular mechanisms underlying
immune responses, inflammation, and the behavior of monocytes/macrophages in
health and disease (Chanput et al., 2014). Researchers use these cells to
investigate signalling pathways, cytokine production, phagocytosis, and the
interaction between immune cells and pathogens or tumor cells.
Lipopolysaccharides (LPS) are large molecules consisting of a lipid and a
polysaccharide portion (Raetz and Whitfield, 2002). They are found in the outer
membrane of Gram-negative bacteria, such as Escherichia coli, Salmonella, and
Pseudomonas species. LPS is an important component of the bacterial cell wall and
plays a crucial role in the structure and integrity of the outer membrane (Aurell and
Wistrom, 1998). LPS is a potent activator of the immune system and is recognized
by various host cell receptors, including toll-like receptor 4 (TLR4) and CD14,
present on immune cells such as macrophages and dendritic cells (Triantafilou and
Triantafilou, 2002). Binding of LPS to these receptors triggers a signalling cascade
that leads to the production of pro-inflammatory cytokines, chemokines, and other
mediators of inflammation. This immune response is an essential part of the host
defence against bacterial infections. However, excessive, or uncontrolled activation
of the immune system by LPS can lead to harmful effects, including septic shock, a
life-threatening condition characterized by systemic inflammation, hypotension, and
multiple organ dysfunction (Pinsky, 2004). LPS-induced septic shock is a significant
cause of mortality in patients with severe bacterial infections. Due to its potent
immunostimulatory properties, LPS is widely used in research laboratories to study
the immune response, inflammation, and the pathogenesis of infectious diseases.
LPS is also used as a vaccine adjuvant and as a model molecule to study host-
pathogen interactions and develop therapeutic strategies targeting the immune
system (van der Poll and Opal, 2008).
Tumor necrosis factor-alpha (TNF-α) is a multifunctional cytokine involved in various
physiological and pathological processes, including inflammation, immune
regulation, cell proliferation, differentiation, and apoptosis (Caminero et al., 2011). It
is primarily produced by activated macrophages, although other cell types such as
lymphocytes, mast cells, endothelial cells, and fibroblasts can also produce TNF-α.
TNF-α plays a critical role in the immune response to infection and in the regulation
of inflammation (Intiso et al., 2004). It stimulates the expression of other cytokines
and chemokines, activates immune cells such as neutrophils and macrophages, and
promotes the migration of immune cells to sites of inflammation. In addition to its role
in the immune system, TNF-α is also implicated in various inflammatory and
autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease,
psoriasis, and ankylosing spondylitis (Toussirot et al., 2012). Drugs that target TNF-
α, such as infliximab, etanercept, and adalimumab, have been developed and are
used in the treatment of these conditions. However, TNF-α can also have detrimental
effects when produced excessively or inappropriately, contributing to the
pathogenesis of conditions such as septic shock, cachexia, and certain
neurodegenerative diseases. Overall, TNF-α is a crucial mediator of inflammation
and immune response, and its dysregulation can have significant implications for
human health and disease.
The induction of TNF-α by LPS is a critical component of the innate immune
response to bacterial pathogens. Studying LPS-induced TNF-α production is
important for understanding the mechanisms underlying the immune response to
bacterial infections and for developing therapeutic strategies to modulate
inflammation in various disease conditions. Researchers often use in vitro cell
culture models and animal models to investigate the signalling pathways involved in
LPS-induced TNF-α production and to evaluate the efficacy of potential anti-
inflammatory agents targeting this pathway.
Several studies have been reported in the literature for the stimulation of THP-1 cells
for the induction of TNF-α. For example, Huang and the group have analysed how
acute psychological stress affects the expression of IL-6 and TNF-α mRNA in
response to LPS stimulation (Huang et al., 2011). To validate the stress model,
measurements were made of heart rate (HR), cortisol, and norepinephrine (NE). The
potential relationships between cytokines and mRNA and other factors (such as age,
BMI, cortisol, and NE) were also investigated. Similarly, TNF-α factor (LITAF) and
obesity and insulin resistance have been sown to be linked (Ji et al., 2011). Their
findings suggested that LITAF may be a novel mediator between inflammation and
diseases associated with obesity. Many other studies have been reported in the
literature for various examinations where LPS induced TNF-alpha have been
involved (Wang et al., 2020, Zhi et al., 2022, Bao et al., 2020).
TNF-α is usually measured using conventional immunoassay techniques (Wang et
al., 2021, Zhi et al., 2022). On the other hand, adapting to the multiple washing
steps, intra- and inter-assay variations, and duration of testing for microtiter plate
experiments may prove difficult in a high throughput setting. When measuring TNF-α
in clinical settings, traditional enzyme-linked immunosorbent technique is unreliable
since large amounts of sample are needed for each assay and bulky, expensive, and
complicated equipment is needed (Bari et al., 2019).

2. Aim

The objective of this research is to guarantee the use of aseptic techniques in the
preparation of incubation, cell culture, culture media, and sample transfer.
Furthermore, the assessment of cell viability and TNF-alpha quantification using
ELISA are examined. The idea that stimulation with lipopolysaccharide (LPS) causes
THP-1 cells to produce more TNF-α is investigated in this work. By conducting these
studies, we hope to further our understanding of how the immune system reacts to
bacterial infections and possibly pinpoint targets for anti-inflammatory drugs.

3. Materials and Methods

3.1 Reagents

 Lipopolysaccharide stock (1000 ng/ml)

 Glutamine
 Penicillin / streptomycin solution
  Foetal calf serum (FCS)
 Trypan blue
 Primary antibody to TNF α in coating buffer
 Wash buffer (phosphate buffered saline- PBS + 0.1% Tween 20)
 Blocking solution (PBS / 2% bovine serum albumin-BSA)
 TNF α Standard (500 pg/ml)
 Conjugated(Biotinylated)Antibody(Ab)
 Streptavidin-HRP (Horseradish Peroxidase)
 Substrate (3, 3', 5, 5'-tetramethylbenzidine or TMB)
 Stop solution (12.5% H2SO4)

3.2 Equipment

 Micropipette
 Glass Bottles
 96-well plates
 24-well plates
 Haemocytometer
 96-well plate reader

3.3 Preparation of cell and medium

The THP-1 cells were provided. These cells were cultured in PMA medium for 72
hours. 500 µl of glutamine and 500 µl of penicillin/streptomycin were aseptically
added to RPMI 1640. The mixture of glutamine, penicillin/streptomycin, and RPMI
1640 was tagged after addition of 5 ml of FCS.
To count the cells, a sterile haemocytometer was employed. 100 ml of the THP-1 cell
solution were transferred to a fresh Eppendorf tube, and 100 millilitres of 0.4% trypan
blue were added to the cell suspension. Next, 10 μl of the solution was transferred to
hemacytometer and cells were counted under 10X objective lens under the
microscope using the formula:

Number of cells counted

Concentration of cells = × 104 × dilution
Number of squares
 Total number of cells counted = 63

Number of squares = 4

63
Concentration of cells = × 104 × dilution
4

Cell viability was calculated by counting number of dead cells stained by trypan blue
and live cells which are not stained. Viability was calculated using the following
formula:
Total viable cells
Cell viability= × 100%
Total cell counted
Total number of cells counted = 63
Number of viable cells = 60
Dead cells = 2
60
Cell viability = × 100%
63
= 95.23

3.4 LPS preparation and assay

The initial concentration of the LPS stock was 1000 ng/ml. Lipopolysaccharide (LPS)
stock was diluted in a full medium that included RPMI, glutamine, antibiotics, and
10% FCS. The final concentrations of LPS employed in the cell stimulation assay
were 1, 10, 50, and 100 ng/ml. 500 μl of LPS was added to 500 μl of cells in the 24-
well plate to create the final concentration of LPS used in the experiment. As a
result, the initial concentration of LPS was essentially cut in half, yielding a 1:1
dilution. Thus, the LPS was first produced at twice the required concentration (2X) to
attain the necessary final concentration. Final concentrations of LPS were 200, 100,
20, and 2 ng/ml. Following the preparation of the LPS, 500 µl of sample was added
to each of the 24 well plate's cells. 500 µl of the pure media were given to the control
or non-stimulated cells. The plate was incubated at 37°C with 5% CO 2 for 24 hrs.
The next day, the supernatant was collected and used in an ELISA to quantify TNF
α.
3.5 Preparation of TNF standards

Standard 500 pg/ml stock solution of TNF α standard was prepared. Using serial
dilution different concentrations of standard was prepared in the ELISA plate ranging
from 0 pg/ml, 7.8 pg/ml, 15.6 pg/ml, 31.25 pg/ml, 62.5 pg/ml, 125 pg/ml, 250 pg/ml
and 500 pg/ml. The final volume of standards in each well was 100 µl.
3.6 Preparation of ELISA plate

100 µl of primary antibody in a coating buffer was added to each well of the ELISA
plate and the plate was sealed to prevent evaporation of the sample. The plate
incubated overnight at 4°C. Next day, wells were rinsed with wash buffer followed by
addition of blocking solution. After 15 minutes, blocking solution was removed from
the plate. After addition of standard and LPS treated cells, we incubated for 20
minutes. After that, wash buffer was used to rinse the standards and supernatants
off the plate. After the plate was dry, secondary conjugated antibody was added to
each well and allowed to sit at room temperature for 15 minutes. The same
procedure was used for STREPTAVIDIN-HRP and the substrate, but the substrate
requiring an incubation step of 20 minutes at 37 degrees Celsius. Ultimately, each
well received 100 µl of stop solution, preparing the plate for OD measurement
3.7 Data analysis

Experimental data were analysed using Microsoft Excel 2013. OD measurements

were imported into the software and calculations were done. Mean value, standard
deviation, coefficient of variance, and linear regression were calculated using
standard formula in excel itself. To check the statistically significant, student t-test
was performed, and p values were measured. P-values of less than 0.05 indicated
the results are statistically significant. All the plots were prepared in excel.

4. Results
4.1 Standard calibration curve
Initially, different concentrations of TNF-α were prepared, and OD measurements
were obtained at 405 nm wavelength. Working concentrations values were 0 pg/ml,
7.8 pg/ml, pg/ml, 15.6 pg/ml, 31.25 pg/ml, 62.5 pg/ml, 125 pg/ml, 250 pg/ml, and 500
pg/ml. OD measurements were imported into excels, and a standard deviation and
coefficient of variance was calculated. In addition, linear regression was conducted.
Equation of trend line is y = 0.0019x - 0.0326 with R 2 value of 0.9976. Table 1
represent calculated values and Figure 1 presents standard calibration curve for
TNF- α.

Table 1: OD measurement values and calculated average and standard deviation values for
different concentration of TNF- α at 405 nm.

TNF-α (pg/ml) OD at 405nm (minus mean SD CoV

background)
Reading Reading Reading
1 2 3
0 -0.01182 -0.00892 -0.00752 -0.0094 0.0022 -23.2821
7.8 -0.00602 -0.00562 -0.00602 -0.0059 0.0002 -3.9231
15.6 0.00098 0.00338 0.00038 0.0016 0.0016 100.4716
31.25 0.02148 0.02258 0.02228 0.0221 0.0006 2.5714
62.5 0.07348 0.07148 0.07248 0.0725 0.0010 1.3797
125 0.17948 0.18348 0.18148 0.1815 0.0020 1.1020
250 0.41748 0.43048 0.42348 0.4238 0.0065 1.5352
500 0.93648 0.92448 0.91748 0.9261 0.0096 1.0375

Figure 1 Standard calibration curve for representing OD values at different concentrations of

TNF- α
4.2 TNF-α concentrations in undiluted sample
After plotting calibration curve, experiments were conducted with LPS stimulated cell
sample to examine secretion of TNF-α at various concentration of LPS. Working
concentrations of LPS were 1 ng/ml, 10 ng/ml and 100 ng/ml. Non stimulated cells
were also included to compare the secretion of TNF-α and check the significance of
this work. OD values were measured at 405 nm. Using regression line obtained in
Figure 1, TNF-α concentrations were calculated. Experiments were done in triplicate.
Table 2 represents OD measured values, average TNF-α concentration along with
standard deviation and coefficient of variance. Students t-test was performed to
verify the significance of the results. During the analysis, background was always
subtracted from measured OD values.

Table 2 OD measurement and TNF-α concentrations in undiluted sample

LPS (ng/ml) OD at 405nm (minus TNF-α concentration mean SD CoV

(pg/ml)
background)
Readin Readin Readin
g1 g2 g3
NS - - - 11.568 11.463 10.463 11.164 0.610 5.463
0.01062 0.01082 0.01272 4 2 2 9 0 7
1 - - - 10.568 10.252 10.252 0.315 3.080
0.01252 0.01372 0.01312 4 9.9368 6 6 8 1
10 - - - 15.831 14.936 14.989 15.252 0.502 3.291
0.00252 0.00422 0.00412 6 8 5 6 1 7
50 48.989 46.357 48.989 48.112 1.519 3.157
0.06048 0.05548 0.06048 5 9 5 3 3 9
100 96.884 97.410 94.778 96.357 1.392 1.445
0.15148 0.15248 0.14748 2 5 9 9 5 1
Figure 2 represents plot of measured TNF-α concentrations in pg/ml at various
concentrations of LPS. It is seen that at LPS concentration of 1 ng/ml, TNF-α
concentration is very low, i.e., 10.25 pg/ml. Surprisingly, concentration of TNF-α for
non-stimulated cells is more than the value obtained for LPS stimulated cells at 1
ng/ml, i.e., 11.16 pg/ml. However, with the increasing concentration of LPS,
secretion of TNF-α also increased. For instance, at LPS concentration of 10 ng/ml,
50 ng/ml and 100 ng/ml, concentrations of TNF-α are 15.25, 48.11 and 96.35 pg/ml
respectively. P-values were calculated, and were found to be 0.069, 0.0029, 0.00042
and 0.0000165 for LPS concentration of 1 ng/ml, 10 ng/ml, 50 ng/ml, and 100 ng/ml
respectively. The result shows that for LPS concentration of 1 ng/ml, results were
non-significant.

Figure 2 TNF-α (pg/ml) against LPS treatment (1-100 ng/ml) for non-diluted sample. NS: Non-
stimulated.

4.3 TNF-α concentrations in diluted sample

Similarly, diluted sample was also used to examine secretion of TNS-α at various
concentration of LPS. Concentrations of LPS used were 1 ng/ml, 10 ng/ml and 100
ng/ml. Non stimulated cells were also added along with positive and negative control.
OD values were measured at 405 nm. In this case also, regression line obtained in
Figure 1 was used to obtain TNF-α concentrations. Experiments were done in
 triplicate. Table 3 represents OD measured values, mean TNF-α concentration along
with standard deviation and coefficient of variance. Students t-test was performed to
verify the significance of the results.

Table 3 OD measurement and TNF-α concentrations in diluted sample

LPS OD at 405nm (minus TNF-α concentration (pg/ml) mean SD CoV

(ng/ml background)
) Readin Readin Readin
g1 g2 g3
NS 0.759 1.832
-0.01322 -0.01252 -0.01302 40.8000 42.2737 41.2211 41.4316 1 1
1 0.917 2.118
-0.01232 -0.01152 -0.01222 42.6947 44.3789 42.9053 43.3263 7 0
10 0.729 1.605
-0.01062 -0.01122 -0.01122 46.2737 45.0105 45.0105 45.4316 3 2
50 1.523 2.084
0.00258 0.00248 0.00128 74.0632 73.8526 71.3263 73.0807 0 0
100 110.484 119.536 114.273 114.764 4.546 3.961
0.01988 0.02418 0.02168 2 8 7 9 3 4

a plot of measured TNF-α concentrations for undiluted sample in pg/ml at various

concentrations of LPS. The concentration measured was multiplied by 4 to obtain
real concentration to incorporate dilution factor. It is seen that at LPS concentration
of 1 ng/ml, TNF-α concentration is increased by nearly 4 folds compared to the neat
sample, i.e., 43.32 pg/ml. Concentration of TNF-α for non-stimulated cells is roughly
like the value obtained for LPS stimulated cells at 1 ng/ml, i.e., 41.43 pg/ml and at 10
ng/ml which is 45.43. However, with the increasing concentration of LPS, secretion
of TNF-α also increased. For instance, at LPS concentration of 50 ng/ml and 100
ng/ml, concentrations of TNF-α are 73.08 and 114.76 pg/ml respectively. P-values
were calculated and was found to be 0.0020, 0.018, 0.00041 and 0.00044 for LPS
concentration of 1 ng/ml, 10 ng/ml, 50 ng/ml, and 100 ng/ml respectively.
Figure 3 TNF-α (pg/ml) against LPS treatment (1-100 ng/ml) for diluted sample. NS: Non-
stimulated.

We also measured TNF-α concentration in positive and negative control from the
experiments. For positive control, concentration of TNF-α is 222.84±2.12 pg/ml
whereas the concentration is 10.56±0.33 pg/ml. Figure 4 represents a plot showing
TNF-αconcentration for positive and negative control.

Figure 4 Plot showing TNF-αconcentration for positive and negative control.

5. Discussion
In the present study, we have shown that increased TNF-α levels in stimulated cells
depends in the concentration of LPS used. TNF-α plays a central role in the immune
response to infection and in the regulation of inflammation. It stimulates the
expression of other cytokines and chemokines, activates immune cells such as
neutrophils and macrophages, and promotes the migration of immune cells to sites
of inflammation. Here, we used THP-1 as a model cell, which is a human monocytic
cell line that is commonly used in biomedical research, particularly in studies related
to immunology, inflammation, and cancer. LPS stimulation of THP-1 cells mimics the
response of immune cells to bacterial infections and is commonly used as an in vitro
model to study inflammation, immune response, and the pathogenesis of infectious
diseases.
In this study, we cultured THP-1 cells, prepare various concentrations of TNF-α from
known standards. Similarly, various concentrations of LPS were prepared to
stimulate THP-cells for the secretion of TNF-α. We further used ELISA technique to
measure OD values and standard curve was plotted. Using equation of linear
regression, TNF-α concentrations were quantified from the experimental data.
It was found that for the neat sample, production of TNF-α is much lower compared
to the results from diluted sample. In diluted sample, with the increasing
concentrations of LPS, secretion of TNF-α increased significantly from 10-100 ng/ml
of LPS. Results also show that there is no significant increase in the production of
TNF-α up to 10 ng/ml of LPS. This suggest that lower concentration of LPS does not
stimulate cells properly. However, there is sharp increase in production of TNF-α at
50 and 100 ng/l of LPS concentrations with results being statistically significant. This
suggest that optimum concentration range of LPS for TNF-α secretion falls between
10-50 ng/ml concentration of LPS. Diluted sample seems to be more effective that
neat sample for these experiments.
As for the method, ELISA technique is found to be highly effective as the calibration
curve is obtained with R2 value of 0.997.

6. Conclusion

The assessment of LPS stimulation on TNF alpha release in THP1 cells using ELISA
has shown that it greatly enhances immune response activation. We can conclude
that ELISA method can be used successfully used for quantification of TNF alpha
released in THP1 cells.
LPS stimulation of THP-1 cells mimics the response of immune cells to bacterial
infections and is commonly used as an in vitro model to study inflammation, immune
response, and the pathogenesis of infectious diseases. These cellular responses
provide valuable insights into the molecular mechanisms underlying the host defence
against bacterial pathogens and the dysregulated immune responses associated
with inflammatory diseases.

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