Peprotech cytokine considerations

Peprotech cytokine considerations

Current methods for detecting specific cytokines in individual cells include: ELIspot, in situ hybridization, immunocytochemistry, limiting dilution analysis (LDA), and single-cell PCR, using in situ hybridization and immunohistochemistry to observe cells. Factor protein expression and mRNA expression can recognize Th1 and Th2 cells. This method can obtain strong intracellular signals, but this method is labor-intensive and subjective, and it is difficult to detect large samples, and human visual recognition ability is very large. Limitations, while ELISPOT and single-cell PCR technology are technically strong and labor intensive, and are difficult to widely promote. With the emergence of multi-label and intracellular cytokine labeling flow cytometry, the study of intracellular cytokines has been pushed to a new stage. The following is mainly to introduce intracellular cytokine flow cytometry.

Early cytokine expression and cell function correlation studies were based on specific clones > clonal cell activation. Although studies using T lymphocyte clones > and TH2 (IL-4, IL-5, IL-10), these studies It is difficult to extrapolate because the T cell clone "g clones demonstrate the synthesis of different cytokines, such as the association of TH1 (IL-2, IFN-> clones with T cell function in vivo has not been revealed.

Recently, Jung and Picker used pre-incubation with monensin, PMA, etc., and blocked intracellular Golgi-mediated transport by Brefeldin (BFA) and Monensin to make cytokines aggregate, accumulate, and enhance cytokine signaling. Cytometry detection. Because T lymphocytes produce a small amount of cytokines in nature, it is usually necessary to study the activation of T lymphocytes in vitro. During the in vitro stimulation, the cytokines produced by T lymphocytes have been released, and the intracellular cytokine signal is weak, making it difficult to detect. This method detects multiple cytokines in a single cell and distinguishes subpopulations of cells that express specific cytokines. At the cellular level, this method demonstrates the presence of type 1 and type 2 differentiation in both human and murine lymphocytes, and these differentiations can be reversed when specific cytokines are enhanced. And these studies clearly demonstrate that only activated cell subsets can express cytokines, and resting normal lymphocytes (T, B, NK) cannot secrete cytokines.

Intracellular flow cytometry is the combination of anti-cytokine antibodies and cell surface or intracellular specific subpopulation markers to detect the secretion of cytokines from different cell subpopulations, while using special chemical and antibody selection to ensure resting and cell-free The minimal fluorescent background of the factor-secreting cells. Advantages that are unmatched by other methods:

Fast: Flow-through quantitative detection of intracellular cytokines can be completed in one day, the experimental process takes 6-8 hours, the actual operation time is 1-2 hours, fast and easy;

Convenient: no tissue culture, whole blood analysis, no need to separate PBMCs;

High sensitivity: highly sensitive fluorescent labeling and detection system;

Efficient: two or more cytokines can be detected simultaneously in the same cell, and subtypes of cells secreting cytokines can be distinguished according to the cellular immunophenotype, and multi-parameter correlation analysis is performed;

Safety: Reduce sample handling and bio-source pollution

Analytical conditions close to the organism: The whole blood test retains the cells and the biochemical microenvironment more accurately reflects the internal conditions.

Second, the required instruments

1. Flow-through sample tube and cell culture dish or plate

2 , 25% CO 2 , 37 ° C incubator

3, mixing oscillator

4, centrifuge

5, sampler, Tips

6, flow cytometry

Third, commonly used specimen types and treatment methods

Whole blood: blood collection using heparin sodium anticoagulation, it is not easy to use lithium heparin, EDTA and ACD anticoagulant, blood samples are analyzed within 8 hours, more than 8 hours will lead to loss of activity, generally cytokine positive cells will be reduced 5 %. If it cannot be detected within 8 hours, the vacuum blood collection tube should be placed at room temperature.

Peripheral blood mononuclear cells (PBMCs) in their own plasma: After blood collection using heparin sodium anticoagulation, PBMCs were isolated and analyzed within 24 hours.

Peripheral blood mononuclear cells (PBMCs) in tissue culture medium: PBMCs were isolated by Ficoll, and the cells were resuspended in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) to adjust the cell concentration to 2×. 106 cells/ml.

The cell concentration was adjusted to 2 x 106 cells/mL in fresh medium.

Frozen whole blood and PBMCs: Activated peripheral blood or PBMCs were treated with 1x red blood cell lysate, rinsed with PBS, resuspended in PBS containing 1% BSA and 10% DMSO, and frozen at -70 °C. After dissolving, the cells were placed in a staining tube, and 2 to 3 mL of the washing solution was added, and after centrifugation for 5 minutes, the cells were disrupted and stained with a rupturing agent.

Fourth, the required reagents

1. Fluorescently labeled monoclonal antibody reagent for cell surface staining

Select special surface markers according to experimental needs

CD45 delineates all lymphocytes

CD3 delineated T lymphocytes

CD4 delineated T helper lymphocyte subsets

CD8 delineated T-inhibited lymphocyte subsets

CD19/ or CD20 delineated B lymphocytes

CD56 delineated NK lymphocytes

CD14 circled monocytes

2 , fluorescently labeled cytokine antibody

R&D provides FITC, PE and APC-labeled cytokine antibodies

3 , hemolysin

Hemolysin should be used when testing peripheral blood (R&D catalog number WL1000 for human or WL2000 for mice; eBioscience catalog number 00-4333) to dissolve red blood cells

4 , activator

1 Phorbol 12-Myristate 13 Acetate (PMA) (Alexis, catalog number ALX-445-004-M001)

Adjusted concentration of 0.1 mg/mL in DMSO

L), stored at -20 °C. Do not freeze and melt m parts (20

1:100 dilution of stock solution in sterile non-azide sodium PBS for each experiment

D. PMA final concentration of 25 ng / mL cell suspension

2 Ionomycin (Alexis, catalog number ALX-450-006-M001)

Formulated in ethanol at a concentration of 0.5 mg/mL

-20 ° C storage

1:10 dilution of stock solution in sterile non-azide sodium PBS for each experiment

g/mL cell suspension m3Ionomycin final concentration 1

Staphylococcal enterotoxin B (SEB) (Sigma, Catalog No. S-4881)

Sterile sodium azide-free PBS adjusted to a concentration of 0.5mg/mL

Store at 4°C

g/mL cell suspension mSEB final concentration 10

4 CD3: coated in culture plates to activate undiluted blood cells in the presence of protein transport inhibitors

5 CD28: Accelerate the activation effect of different stimulants (including SEB, CD3, etc.), the concentration is generally 10ug/ml

5 , blocker

Blocking Golgi-mediated transport, allowing cytokines that stimulate cell expression to accumulate in the cytosol

1 Brefeldin-A (BFA) (eBioscience, catalog number 00-4506)

Adjust the concentration of 5mg/mL in DMSO

Dispense (20L), store at -20 °C. Do not freeze and thaw repeatedly.

1:10 dilution of stock solution in sterile non-azide sodium PBS for each experiment

g/mL cell suspension. m activates the final 4-5 hours BFA final concentration 10

Note: excessive BFA incubation leads to decreased cell viability

2 Monensin (eBioscience, catalog number 00-4505)

6 , Glutamine-free RPMI-1640

7 , Fixative

The cells need to be fixed after stimulation in vitro. The purpose of immobilization is to immobilize cytokines in cells on the one hand and cell surface antigens on the other hand through cross-linking and denaturation of proteins. 4% paraformaldehyde in PBS (eBioscience, catalog number 00-8222)

8 , Breaking agent

Hanks' solution (HBBS) containing 1% saponin, 0.05% sodium azide (eBioscience, catalog number 00-8333)

Perforation of the cell membrane has facilitated the entry of fluorescently labeled cytokine antibodies into the cell for binding of the corresponding cytokines

9 , other reagents

Sterile sodium azide free PBS, ethanol, PBS containing 1% paraformaldehyde, stored at 4 °C.

Fifth, the basic method of cell culture and stimulation

The ultimate result of cell activation is the production of cytokines. Depending on the test and the specimen, the investigator needs to select different stimulators and stimulation times to get the best results. The table below provides recommended activation methods for detecting some common cytokines.

Table 1. Recommended positive control activation method for intracellular cytokine flow detection

Cytokine

Positive control stimulation method

Human IFN-g

Method 2 (4-24 hours)

Human TIMP-1

Method 5

Human aTNF-

Method 7 (6 hours)

Human IL-1a

Method 3 (6 hours)

Human bIL-1

Method 3 (24 hours)

Human IL-2

Method 2 (4-24 hours)

Human IL-4

Method 4

Human IL-5

method 1

Human IL-6

Monocytes: Method 3 (6-12 hours) T cells: Method 6

Human IL-10

method 1

Human IL-12

Method 8

Human IL-15

Method 3

Human Fractalkine/CX3CL1

method 1

Human IL-8/CXCL8

Method 3 (24 hours)

Human MCP-1/CCL2

Method 3 (24 hours)

Human MIP-1a/CCL3

Method 3 (24 hours)

Human MIP-1b/CCL4

Method 3 (24 hours)

Human RANTES/CCL5

method 1

Mouse IL-2

Method 9

Mouse IL-4

Method 10

Mouse IL-5

Method 10

Mouse IL-6

Method 11

Mouse gIFN

Method 9 or Method 12

Mouse aTNF-

Method 9

To prevent the secretion of intracellular cytokines into the extracellular space, protein transport inhibitors (eg 3uM monensin, 10 mg/ml BFA) are required during the last 4-6 hours of culture.

Method 1: Use only transfected cells for detection

Method 2: Human PBMCs were stimulated with PMA (10 ng/ml) and Ionomycin (1 uM) for 4-24 hours.

Method 3: Human PBMC were stimulated with LPS (0.5 - 1 ug/ml) for 24 hours.

Method 4: Human PBMCs or purified CD4+ cells in a culture plate coated with human CD3 antibody, using recombinant human IL-2 (10 ng/ml, catalog number 202-IL-010) and IL-4 (10) The medium of ng/ml, catalog number 204-IL-005) was cultured for two days; after cell washing, the culture was continued for 2 days in a medium containing recombinant human IL-2 and IL-4; finally, the cells were harvested using PMA (10). Ng/ml) and Ionomycin (1 uM) were stimulated for 6 hours in 20 mL in a Falcon tube, and 100 mL of activated whole blood (cell concentration maintained at 2×10 6 /mL) was added and mixed, and incubated for 15 minutes at room temperature in the dark;

Method 5: CD4 T cells stimulated with PHA (10 ng/ml) for 4 days

Method 6: T cells use anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody and recombinant human IL-1b (Lorre, et al., 1994, Clin Immuno Immunopath 70:1)

Method 7: Stimulate with PMA (50 ng/ml) and Ionomycin (500 ng/ml)

(10 ng/ml, catalog number 285-IF-100) Stimulate for 2 hours, then stimulate with IFN (10 ng/ml) LPS (1 ug/ml) for 22 hours or use the same method to stimulate THP-1 cells. 8: PBMCs cells use recombinant human IFN

Method 9: EL4 was stimulated with anti-CD28 antibody (2 ug/ml) PMA (5 ng/ml) Ionomycin (500 ng/ml) for 6 hours in a plate coated with anti-mouse CD3 antibody (25 ug/ml).

Method 10: CD4+ T cells Recombinant mouse IL-2 (10 ng/ml in a culture plate coated with mouse CD3 (25 ug/ml) antibody using an antibody containing anti-mouse CD28 (2 ug/ml) , catalog number 402-ML-020) and IL-4 (50 ng/ml, catalog number 404-ML-005) culture medium for two days; then in medium containing recombinant mouse IL-2 and IL-4 The culture was continued for 3 days; cells were finally harvested and stimulated with PMA (5 ng/ml), Ionomycin (500 ng/ml) and monensin for 6 hours.

Method 11: Mouse ip macrophages were stimulated with Mouse ip 1 ug/ml LPS for 24 hours.

Method 12: Mouse spleen cells were stimulated with PMA (5 ng/ml) and Ionomycin (500 ng/ml) for 6 hours.

Sixth, the basic process of flow detection cell staining (taking whole blood as an example)

1 , harvesting cells

Heparin sodium anticoagulated venous blood, mixed with 1:1 medium, add stimulant, and add protein transport inhibitor (refer to the instructions), mix, 37 ° C, 5% carbon dioxide culture 4-6 hours

2 , blocking Fc receptor

Used to eliminate non-specific binding staining

1 In mice, purified FcII/III receptor antibody (CD16/32) can be used for 15 minutes in staining buffer at 4 °C for 1 ug/106 cells. After washing with PBS, the next step is directly stained. .

2 For humans and rats, excessive use of purified Ig or serum that is not related to the same source and subtype of fluorescent antibody can be used directly to block

3 , cell surface staining

1 plus appropriate cell surface staining reagent

2 Add hemolysin and incubate for 10 minutes in the dark at room temperature. (Note: PMA-activated whole blood may be incompletely hemolyzed.)

3 Centrifuge 500g for 5 minutes, discard the supernatant

4 , fixed and ruptured

1 Add a fixative, incubate for 15 minutes at room temperature in the dark, centrifuge 500g for 5 minutes, discard the supernatant;

2 Incubate the membrane in a warm and dark place for 10 minutes. (Dose reference manual)

Wash the solution in 3 mL of PBS, centrifuge 500 g for 5 minutes, and discard the supernatant. ~3 plus 2

5 , intracellular staining

1 Add fluorescently labeled cytokine antibody, mix and incubate for 30 minutes at room temperature in the dark.

LPBS on the machine or add 500 L 1% PFA fixed, then add 2, 3mL washing solution on the machine m2, centrifuge 500g for 5 minutes, discard the supernatant, add 500

Seven, matters needing attention

1 , specimen processing

Avoid the use of complexed calcium anticoagulants such as ACD and EDTA, as they limit the calcium-dependent activation process, and heparin sodium is recommended. In addition, LPS, a common biological agent contaminant, is a strong cell activator that may confuse test results. Blood samples were analyzed within 8 hours, and more than 8 hours resulted in loss of activity, and cytokine positive cells were generally reduced by 5%. If it cannot be detected within 8 hours, the vacuum blood collection tube should be placed at room temperature.

2 , stimulate activation

Detect different cytokines Select different stimulator combinations and stimulation times according to the situation to ensure the best detection results. For example, to detect IFN-γ, PMA and Ionomycin can be stimulated simultaneously; if CD4 is used for surface labeling, since most patients' CD4 antigens will be down-regulated due to PMA activation, the culture time should not be too long. 4- 6 hours is appropriate, otherwise the impact of CD4 down-regulation

3 , choose the appropriate control

To ensure the trueness and reliability of the combination, at least the following settings for fluorescence:

1 unstimulated control

When activated, the presence of BFA inhibits the transport of intracellular proteins, so antigens and cytokines produced during activation are retained in the cells, and unstimulated controls should also contain BFA.

2 activation control

Activation of the control using cell surface expression of CD69 to assess activation or not, if the desired level of CD69 is not reached, the activation step is problematic, an agent may be inactivated, expired, improperly prepared or solvent contaminated, and a fresh stimulant is required to retest .

3 isotype control

Immunoglobulins of the same source, identical label, identical dose and subtype as the fluorescently labeled antibody are used to eliminate background staining due to non-specific binding of the antibody to the cell surface.

4 , Fc receptor blockade

Blocking Fc receptors with reagents can effectively reduce non-specific fluorescent staining. Purified anti-mouse CD16/32 (eBioscience, catalog number 14-0161-81) can be used in mice, and purified anti-large in rats. In mouse CD32, Ig ​​or serum can be purified in humans with an excess of the same species.

5 , the choice of fluorescein

PE or APC labeling should be used when detecting relatively low expression of cytokines such as IL-4; PE or APC labeling should also be used when detecting a single cytokine; when detecting multiple cytokines, weakly expressed PE or APC, FITC labeling is best used for high expression of cytokines such as IFN-[gamma].

Eight, questions and answers

problem

the reason

solve

Comment

No CD69 intracellular staining

Cell not activated

Improper preparation of the activator, see the Activator Preparation Storage section.

The CD69 positive rate of CD3 T lymphocytes should be >90% after 4 hours of PMA Ionomycin activation.

Wrong anticoagulant used

Heparin sodium should be used, do not use lithium heparin, and avoid the use of complex anticoagulants such as ACD and EDTA.

Lymphocyte activation requires Ca, and the anticoagulant that complexes Ca affects activation.

Cells are not transparent

Use hemolysin before using the permeate

Hemolysin-assisted cells are transparent

BFA is inactivated or improperly prepared

See BFA Preparation BFA for storage at -20 ° C for details.

See BFA preparation for details.

Intracellular staining is positive but weak

Anti-cytokine antibody concentration is wrong

Use fluorescent antibodies as recommended by R&D

Normal activation T

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