The effect of drugs on macrophages in the peritoneal cavity of animals was experimentally studied by colorimetric and fluorescent staining and flow cytometry.
Operation method
flow cytometry
Principle
Rhodiola rosea glycosides not only have a variety of pharmacological effects such as anti-aging, antidepressant, anticancer, anti-hypoxia, liver protection, improving cardiovascular and cerebral vascular function and enhancing central nervous system function, but also play a role in the pharmacological effects of anti-fatigue, anti-hypoxia, anti-tumor, anti-virus and enhancement of immune function. Macrophage is an important presenting cell of the immune system and plays an important role in both specific and non-specific immune processes.
Materials and Instruments
Mouse Move I. Preparation of materials1. Preparation of mouse peritoneal macrophage suspensions Common Problems I. Discussion The immunoregulatory role of macrophages is realized through their antigen presentation, phagocytosis and secretion of various cytokines. The basis of macrophage immune function is cell survival, and its survival status directly affects its function and the strength of response. For more product details, please visit Aladdin Scientific website.
Sal Sword Bean Protein A Tetramethyl azole L-glutamine β-Dimercaptoethanol Actinidione Penicillin Streptomycin RPM II 1640 Medium Fetal Bovine Serum Carboxyfluorescein bis(acetate) Succinimidyl Ester 2-7-Dichlorohydrofluorescein ethylenedibasic G riess Kit
Enzyme Labeler Flow Cytometer Fluorescein Labeler
(1) The eyes of BALB/c mice were bled to death, and the skin of mice was sterilized with 750 mL/L ethanol.
(2) 5 mL of RPMI1640 basal medium was injected intraperitoneally.
(3) Gently rub the abdomen of mice for 5 min, carefully aspirate the liquid from the lateral abdominal wall, collect the cells into a 15 mL centrifuge tube, centrifuge the cells at 1500 r/min for 5 min at 4 ℃, discard the supernatant, resuspend the cells with RPMI1640 basal medium, and adjust the cell density to 2×109 cells/L, inoculate the cells into 96/24-well cell culture plates, and then incubate the cells in a 50 mL/L CO2 incubator at 37 ℃. CO2 incubator.
(4) After 3 h, the upper layer of culture medium was discarded and washed gently with cold RPMI1640 basal medium for two times to remove the unaffixed cells to obtain the affixed macrophages.
II. Experimental steps
1. Control experiment
(1) A blank control group (with DMEM complete culture medium), a control group (with macrophage suspension), a Sal80 μmol/L group, a Sal160 μmol/L group and a Sal320 μmol/L group were set up in the experiment, and three wells were set up in each group.
(2) Peritoneal macrophages with a density of 2×109 cells/L were inoculated in 96-well plates, and 190 μL of RPMI1640 basal medium was added to each well, and 10 μL of Sal working solution was added to the final concentrations of 80 μmol/L, 160 μmol/L and 320 μmol/L, respectively, and the cells were cultured in a 50 mL/L CO2 incubator at 37℃ for 48 hours. for 48 h. The incubation was performed at 37℃ in a 50 mL/L CO2 incubator.
(3) Add 20 μL of MTT (5 mg/L) to each well, incubate for 4 h, centrifuge (1500 r/min, 5 min) and discard the supernatant, then add 100 μL of Dimethyl Sulfoxide (DMSO) to each well, and vibrate for 10 min in a shaker, avoiding light, in order to dissolve the purple crystals at the bottom of the wells, and then measure the absorbance value of each well in an enzyme counter at 490 nm, and calculate the relative survival rate of cells. The relative cell survival rate was calculated.
(4) Relative cell survival rate = [(A value of Sal group at different concentrations - A value of blank control group)/(A value of control group - A value of blank control group)] × 100%.
2. Detection by MTT colorimetric method
(1) The experiment was set up with control group, LPS+IFN-γ group and Sal+LPS+IFN-γ group, and each group was set up with 3 replicate wells.
(2) Peritoneal macrophages with a density of 2×109 cells/L were inoculated into 96-well plates, and 190 μL of RPMI1640 basal medium was added to each well, and 10 μL of Sal at different concentrations (final concentrations of 80, 160, and 320 μmol/L) was added and incubated for 4 h. The cells were then incubated for 4 h with the addition of 10 μL of Sal at different concentrations (final concentrations of 80, 160, and 320 μmol/L).
(3) LPS9 (final concentration of 10 mg/L) and IFN-γ (final concentration of 80 μg/L) were added and incubated for 48 h at 37°C in a 50 mL/L CO2 incubator.
(4) 20 μL of MTT (final concentration of 5 mg/L) was added to each well, and the incubation was continued for 4 h before centrifugation (1500 r/min, 5 min).
(5) Discard the supernatant, add 100 μL of DMSO to each well, shake for 10 min, and detect the A value at 490 nm with an enzyme marker, and calculate the proliferation promotion rate of Sal on macrophages.
(6) Promotion rate = [(A value of Sal+LPS+IFN-γ group - A value of LPS+IFN-γ group)/A value of LPS+IFN-γ group]×100%.
3. Detection by SytoxGreen staining method
(1) The experiment was set up with control, CHX and Sal+CHX groups, and 3 replicate wells were set up in each group.
(2) Peritoneal macrophages with a density of 2×109 cells/L were inoculated into 96-well plates, 190 μL of RPMI1640 basal medium was added, and 10 μL of Sal working solution with a final concentration of 160 μmol/L was added to the adherent macrophages.
(3) After 4 h of incubation, CHX was added to the CHX group at a final concentration of 0.5 μmol/L, and incubation was continued for 48 h at 37°C in a 50 mL/L CO2 incubator.
(4) Macrophages in 96-well plates were stained with Sytox ?Green at a final concentration of 0.5 μmol/L for 15 min at room temperature, and the fluorescence intensity of Sytox Green staining of macrophages was detected by a fluorochrome marker.
4. Detection by immunofluorescence microsphere assay
(1) The experiment was set up with a control group, LPS+IFN-γ group, Sal group with different concentrations and Sal+LPS+IFN-γ group, and 3 replicate wells were set up in each group.
(2) Peritoneal macrophages with a density of 2×109 cells/L were inoculated into 24-well culture plates, and 500 μL of RPMI1640 basal medium was added to each well, and 10 μL of different concentrations of Sal (final concentrations of 80, 160, and 320 μmol/L) were added.
(3) 37℃, 50 mL/L CO2 incubator for 4 h. The incubation was carried out in the incubator.
(4) Add LPS at a final concentration of 10 mg/L and IFN-γ at a final concentration of 80 μg/L, and incubate at 37℃, 50 mL/L CO2 incubator for 24 h. Finally, add fluorescent microspheres with a diameter of 1 μm (at a final concentration of 1×1010/L ), and continue to incubate for 2 h. The final concentration of IFN-γ was 0.5 μm.
(5) The supernatant was gently aspirated off, and the unphagocytosed microspheres were gently washed with PBS to remove the unphagocytosed microspheres, and then the adherent abdominal macrophages were eliminated with trypsin.
(6) Cells were collected, centrifuged (1500 r/min, 5 min), and resuspended in 200 μL PBS and immediately assayed by FCM.
5. Detection by H2DCFDA staining method
(1) The experiment was set up with a control group, LPS+IFN-γ group and Sal+LPS+IFN-γ group, with 3 replicate wells in each group.
(2) Peritoneal macrophages were inoculated into 96-well culture plates at a density of 2×109 cells/L. 190 μL of RPMI1640 basal medium was added to each well, and 10 μL of Sal at different concentrations (final concentrations of 80, 160, and 320 μmol/L) was added.
(3) The incubator was incubated at 37°C for 4 h with 50 mL/L CO2.
(4) Add LPS at a final concentration of 10 mg/L and IFN-γ at a final concentration of 80 μg/L, and incubate for 24 h at 37°C, 50 mL/L CO2 incubator.
(5) Discard the supernatant, and stain the wells with H2DCFDA staining solution at a final concentration of 5 μmol/L, and add 100 μL of staining solution to each well for 30 min at room temperature, protected from light.
(6) Detection was carried out by a fluorescence enzyme marker with an emission wavelength of 485 nm and a detection wavelength of 520 nm.
6. Detection using the Griess kit method
(1) The experiment was set up with a control group, LPS+IFN-γ group and Sal+LPS+IFN-γ group, and peritoneal macrophages with a density of 2×109 cells/L were inoculated into 24-well culture plates.
(2) Each well was spiked with 500 μL of RPMI1640 basal medium, and 10 μL of Sal at different concentrations (final concentrations of 80, 160, and 320 μmol/L) was added.
(3) Incubate for 4 h at 37℃ and 50 mL/L CO2 incubator.
(4) Add LPS at a final concentration of 10 mg/L and IFN-γ at a final concentration of 80 g/L.
(5) Incubate at 37℃, 50 mL/L CO2 incubator for 24 h. Aspirate 50 μL of supernatant into 96 cell culture plates, and test according to the instructions of the Griess kit, and detect the A value at a wavelength of 540 nm with an enzyme labeler.
(6) At the same time, the standard curve was plotted according to the instructions of Griess kit and the amount of NO produced in each group was calculated.
7. Statistical analysis: The results were expressed as x±s, and n was the sample size. Statistical graphing software was used in Microsoft Excel, and group comparisons were made using the One-way t-test, with P<0.05 being statistically significant.
The protective effect on cell survival is generally accomplished in two ways: one is to promote cell growth, and the other is to inhibit apoptosis. For this reason, a series of experiments were designed for macrophages in this experiment, and it was found that Sal could promote the proliferative response of mouse peritoneal macrophages stimulated by LPS, suggesting that Sal may act as a secondary signaling to LPS and IFN-γ to promote the signaling and cytokine secretion of macrophages in a synergistic manner, thus promoting their proliferation. It is therefore suggested that Sal may have an enhancing effect on intrinsic immunity in mice.
The results of this experiment showed that Sal had an inhibitory effect on CHX-induced apoptosis. CHX is known to be an inhibitor of protein synthesis, which can inhibit protein synthesis by directly binding to protein translocase and interfering with the transcription process. Therefore, we hypothesized that Sal might block the binding of CHX to protein translocase through certain pathways and thus inhibit CHX-induced apoptosis.
From the results of the above two experiments, we can conclude that Sal has a protective effect on the survival of mouse peritoneal macrophages. Phagocytosis is one of the important functions of macrophages, which improve the body's anti-infection ability by phagocytosis of invading pathogens and senescent and aberrant cells in the body.
Macrophage recognition of antigenic foreign substances such as pathogens is through its surface patternrecognitionreceptor (PRR) to directly recognize the highly conserved specific molecular structures shared by certain pathogens or their products, or through the surface IgGFc receptor (Fc??R) and complement receptor (CR) to recognize IgG or complement-bound Pathogen.
Macrophages bind to antigenic foreign bodies such as pathogens and then ingest them intracellularly through phagocytosis or phagocytosis to form phagosomes. Within the phagosome, pathogens can be killed by oxygen-dependent and oxygen-independent bactericidal systems. We investigated the effect of Sal on macrophage phagocytosis, and the results showed that Sal could significantly improve the phagocytosis function of macrophages in both resting and activated states, and it was hypothesized that Sal might promote the combination of PRR on the surface of macrophages and specific molecular structures on the surface of pathogens or apoptotic cells through a certain molecular structure or biological mechanism, thus promoting the phagocytosis function of macrophages, and enhancing the macrophages' intrinsic immune response to foreign pathogens.
Ryter et al. showed that ROS are able to initiate an apoptotic cascade response in cells. The production of excess intracellular ROS is not only associated with apoptosis and necrosis, but also with many hypoxia-associated pathologies (e.g., local ischemic injury and tumors).
Both clinical and in vivo experiments have shown that Rhodiola rosea has a protective effect against local ischemic injury. The results of this study now indicate that Sal is the main active component of Rhodiola rosea and can have a protective effect against apoptosis induced by excessive intracellular ROS production caused by hypoxia.Sal scavenges different types of superoxide anion radicals and hydroxyl radicals in the organism.Therefore, the effect of Sal on LPS and IFN-γ-induced intracellular ROS production by peritoneal macrophages was investigated in this experiment.
The results showed that Sal significantly reduced intracellular ROS in activated macrophages, suggesting that the protective mechanism of Sal may be the direct scavenging of intracellular ROS in peritoneal macrophages, thereby protecting against apoptosis induced by excess ROS in peritoneal macrophages.
NO, as a nonspecific molecule secreted by activated macrophages, can kill or inhibit the growth of a wide range of pathogenic microorganisms. Macrophage NO is synthesized via the NOS pathway, and its synthesis capacity is closely related to its anti-inflammatory effects.NO synthesis can be induced by IFN-γ, however, this induction process usually requires the presence of a second signal (TNF- and different microbial components such as LPS).IFN-γ or the second signaling alone induces macrophages to synthesize a low amount of NO but, in many cases, has no direct cytotoxic effect; however, IFN-γ and the second signaling together can significantly increase the synthesis of NO by activated macrophages. cytotoxic effects; however, IFN-γ and second signaling, when acting together, significantly increase NO production and increase cytotoxic effects in macrophages.
Xeres et al. demonstrated that peritoneal macrophages from Sal-immunized mice significantly increased the ability to kill HCa-F hepatocellular carcinoma cells, and it was speculated that Sal may exert its tumor-killing effect through iNOS-mediated NO synthesis.
Seo et al. showed that Sal promoted transcription and expression of iNOS genes in IFN-γ-activated RAW264.7, but Sal alone had no effect on iNOS gene expression when acting on resting RAW264.7.
We investigated the effect of Sal on NO secretion by peritoneal macrophages. The results showed that Sal had a synergistic effect on NO secretion from LPS- and IFN-γ-induced activated peritoneal macrophages, suggesting that Sal may be acting as a secondary signal to promote iNOS genes in peritoneal macrophages
In conclusion, Sal can enhance the phagocytosis function of macrophages in both resting and activated states, and at the same time promote the secretion of NO and reduce the production of intracellular ROS in macrophages, with little toxic side effects, suggesting that it can improve the function of the whole body's intrinsic immunity, and has the potential to become an adjunctive therapeutic drug for tumors, infections, autoimmune diseases, immunodeficiency diseases, etc., and a promising immunomodulatory agent.