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STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00432-5
10.1016/j.xpro.2024.103267
103267
Protocol
Protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages
Xu Jin-Chuan 12
Hu Zhidong huzhidong@fudan.edu.cn
1∗
Fan Xiao-Yong xyfan008@fudan.edu.cn
13∗∗
1 Shanghai Institute of Infectious Diseases and Biosecurity & Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
∗ Corresponding author huzhidong@fudan.edu.cn
∗∗ Corresponding author xyfan008@fudan.edu.cn
2 Technical contact

3 Lead contact

17 8 2024
20 9 2024
17 8 2024
5 3 103267© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Bacillus Calmette-Guérin (BCG), the only licensed tuberculosis vaccine, provides non-specific protection against non-tuberculosis diseases that is mediated by trained immunity, a functional reprogramming mediated by innate immune memory. Here, we present a protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages (BMDMs). We describe steps for preparing BCG single bacterial suspensions, isolating BMDM cells, and the training process. This protocol can assist researchers to conveniently utilize BMDM cells to study trained immunity.

For complete details on the use and execution of this protocol, please refer to Xu et al.1

Graphical abstract

Highlights

• Differentiation and culture of murine BMDM

• Preparation of BCG single bacterial suspensions by low-speed centrifugation

• In vitro induction of trained immunity in BMDM cells

• Determination of trained immunity by assessing cytokine production levels

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Bacillus Calmette-Guérin (BCG), the only licensed tuberculosis vaccine, provides non-specific protection against non-tuberculosis diseases that is mediated by trained immunity, a functional reprogramming mediated by innate immune memory. Here, we present a protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages (BMDMs). We describe steps for preparing BCG single bacterial suspensions, isolating BMDM cells, and the training process. This protocol can assist researchers to conveniently utilize BMDM cells to study trained immunity.

Subject areas

Cell culture
Cell isolation
Immunology
Microbiology
Model Organisms
==== Body
pmcBefore you begin

Bacillus Calmette-Guérin (BCG), the only licensed tuberculosis (TB) vaccine, has been widely used worldwide to prevent TB since it was first administered to humans in 1921.2 It has been discovered that the BCG vaccination provides non-specific protection against cancers, autoimmune disorders, and other infectious diseases in the last century.3 Intravesical instillation of BCG for the treatment of bladder cancer is undoubtedly the most successful application of BCG for non-specific protection.4 Recently, it has been found that BCG can enhance the response of innate immune cells (e.g., macrophages) to secondary stimuli by reprogramming their metabolism and epigenetic modifications, a phenomenon that has been named “trained immunity”.5 Trained immunity is thought to be one of the mechanisms of BCG-induced nonspecific protection,6 and the activation of glycolysis and glutamine metabolism in BCG-trained macrophages is critical for functional remodeling.7 Recently, we compared differences in the ability of different BCG strains to induce trained immunity by constructing in vivo and in vitro models.1 Here, we will detail the model of BCG-induced trained immunity of BMDM cells.

Optimization of BCG single bacterial suspension preparation

Since mycobacterium cell walls have a rich lipid layer, they tend to agglomerate in polar media (e.g., PBST) (Figure 1A), resulting in inaccurate quantification. BCG should be prepared as a single bacterial suspension to be accurately quantified. BCG-EGFP, a BCG strain expressing a green fluorescent protein, was used in this protocol to directly evaluate the preparation of single bacterial suspensions through a fluorescence microscope. However, other researchers could also use wild-type BCG to follow this protocol. The BCG-EGFP was grown at 37°C in liquid Middlebrook 7H9 broth (BD Difco, USA) supplemented with 10% (v/v) oleic acid-albumin-dextrose-catalase enrichment (OADC; BD Difco, USA), 0.5% glycerol, 0.05% Tween-80 and 50 μg/mL hygromycin B. PBST was used to prepare BCG single bacterial suspensions.Figure 1 Optimization of single bacterial preparation conditions

(A) BCG suspension after resuspension with PBST. 2 μL of the suspension is taken onto a slide, then covered with a cover glass and observed under a microscope (scale 100 μm).

(B) BCG suspension after low-speed centrifugation (scale 100 μm).

(C) OD600 values and loss rates of BCG suspensions after centrifugation at different centrifugal forces for 2 min Loss rate = 100 - post-centrifugation OD600/pre-centrifugation OD600 × 100.

(D) BCG suspensions centrifuged for 2 min at 100× g. White arrows refer to BCG clumps (scale 50 μm).

(E) BCG suspensions centrifuged for 2 min at 300× g (scale 50 μm).

(F) BCG suspensions centrifuged for 2 min at 500× g (scale 50 μm).

(G) OD600 values and loss rates of BCG suspensions at different times of centrifugation at 500× g.

(H) Proportion of different sizes of bacterial clump size by centrifugation at 500× g for 1 min and 2 min, respectively. Two-way ANOVA test was used to compare groups: ∗ P < 0.05. ∗∗∗ P < 0.001. Values are expressed as mean ± SD.

(I) Single bacterial suspension obtained by centrifugation at 500× g for 2 min (scale 100 μm).

This study used low-speed centrifugation to prepare BCG single bacterial suspensions. Initially, low-speed centrifugation at 200× g for 5 min yielded single bacteria, indicating that low-speed centrifugation is a method worthy of continued optimization (Figure 1B). We next evaluated the loss rates of the single bacterial suspension preparation methods at different relative centrifugation forces after 2 min of centrifugation. The loss rates at 800× g and 1000× g were both greater than 70% (Figure 1C), whereas the single bacterial suspensions obtained at 100× g (Figure 1D) and 300× g (Figure 1E) still had small clumps, with the fewest clumps at 500× g (Figure 1F). Thus, centrifugation time was subsequently optimized at 500× g. The loss rate by centrifugation for 3 min was greater than 70% (Figure 1G), and centrifugation for 2 min resulted in a significantly higher proportion of single bacteria (∼80%) than the 1 min group (∼70%) (Figure 1H). In summary, centrifugation at 500× g for 2 min could stably and rapidly prepare fresh single bacterial suspensions needed for subsequent experiments (Figure 1I).Note: Centrifugation in this protocol was all performed at room temperature with an acceleration and deceleration of 9.

Institutional permissions

All experiments were performed in accordance with the institutional guidelines of the Animal Research and Ethics Committee of Shanghai Public Health Clinical Center.

General laboratory preparations prior to starting the work

All operations are performed in a Class I biosafety cabinet using the standard aseptic technique, and cells are incubated at 37°C in a humidified incubator with 5% CO2.1. Make sure all reagents are available and sterile.

2. Start the cell culture laminar flow bench and carefully sterilize surfaces.

3. Set the water bath at 37°C and prewarm medium.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Chemicals, peptides, and recombinant proteins	
	
Fetal bovine serum (FBS)	Gibco	16000-044	
Red blood cell lysis buffer	Beyotime	C3702-120 mL	
Phosphate-buffered saline (PBS)	Absin	Abs962	
Gentamycin	Sangon Biotech	A506614-0005	
Dulbecco’s modified Eagle’s medium/nutrient mixture F12 Ham (DMEM/F12)	VivaCell Biosciences	C3130-0500	
Recombinant murine M-CSF	PeproTech	Z02930	
0.25% trypsin-EDTA	NCM Biotech	C100C1	
Middlebrook 7H9 broth	BD Difco	271310	
Middlebrook oleic albumin dextrose catalase supplement (OADC)	BD Difco	212351	
Tween 80	Sangon Biotech	A100442-0500	
Glycerin	Sangon Biotech	A501745-0500	
Lipopolysaccharide (LPS)	Beyotime	S1732-0.5mg	
Hygromycin B	Sangon Biotech	A600230-0001	
	
Critical commercial assays	
	
TNF-α mouse ELISA kit	Invitrogen	88-7324-88	
IL-6 mouse ELISA kit	Invitrogen	88-7064-88	
IL-1β mouse ELISA kit	Invitrogen	88-7013A-88	
	
Other	
	
Insulin syringe	Kindly Medical Instruments	C20230920	
10 cm cell culture-treated dish	JET BIOFIL	TCD010100	
48-well cell culture plate	YUEYIBIO	20230420	
Culture square flask (30 mL)	SORFA	277110	
0.22 μm syringe filter	JET BIOFIL	FPE204030	
70 μm cell strainer	Falcon	352350	

Materials and equipment

BCG-EGFP was constructed by electroporating BCG Pasteur (a gift from Prof. Xiao-ming Zhang, Pasteur Institute of Shanghai, Chinese Academy of Sciences) with a plasmid expressing EGFP (i.e., EGFP was cloned into the pMFA41 plasmid) and screened with kanamycin (final concentration: 50 μg/mL) on Middlebrook 7H11 agar.8Cell culture medium

Reagent	Final concentration	Amount	
DMEM/F12	90%	450 mL	
FBS	10%	50 mL	

Note: Store at 4°C for up to six months and warm up to 37°C prior to use.

Mycobacteria culture medium

Reagent	Final concentration	Amount	
Middlebrook 7H9 Broth	N/A	4.7 g	
OADC	10%	100 mL	
ddH2O	90%	900 mL	
10% Tween 80	0.05%	5 mL	
40% Glycerin	0.5%	12.5 mL	

Note: Store at 4°C for up to six months.

40% Glycerin

Reagent	Final concentration	Amount	
Glycerin	40%	40 mL	
ddH2O	60%	60 mL	

Note: Store at 4°C for up to twelve months.

10% Tween 80

Reagent	Final concentration	Amount	
Tween 80	10%	10 mL	
ddH2O	90%	90 mL	

Note: Store at 4°C for up to twelve months.

0.05% PBST

Reagent	Final concentration	Amount	
PBS	N/A	1000 mL	
Tween 80	0.05%	0.5 mL	

Note: Store at 4°C for up to twelve months.

Note: The mycobacteria culture medium, 40% glycerol, 10% Tween 80 and 0.05% PST are all sterilized by passing them through a 0.22 μm syringe filter.

CRITICAL: Medium should be prepared sterilely in a biosafety cabinet.

Alternatives: No key materials/equipment that cannot be substituted.

Step-by-step method details

BCG resuscitation and culture

Day −3

Timing: 10 min

1. Take 100 μL of BCG-EGFP strain (OD600 ∼ 0.5) stored at −80°C into a culture square flask containing 5 mL of fresh mycobacteria culture medium with 50 μg/mL kanamycin and incubate in a shaker at 37°C, 100 revolutions per min (rpm).

Note: To store BCG strains, logarithmic growth phase BCG cultures were mixed 1:1 with 40% glycerol and frozen at −80°C.

Day 0

Timing: 10 min

2. Measure the OD600 of BCG-EGFP cultures (First passage, OD600 ∼ 0.5).

3. Discard 3.5 mL of culture and supplement with 3.5 mL of fresh mycobacteria culture medium containing 50 μg/mL kanamycin (OD600 ∼ 0.2).

4. Incubate in a shaker at 37°C, 100 rpm.

Preparation and culture of BMDM from mice

Day 1

Timing: 90 min

5. Sacrifice specific pathogen-free (SPF) grade BALB/c mouse and remove the hind legs. Sterilize and dehydrate the hind legs by soaking in 75% alcohol for 5 min (Figure 2A).Figure 2 Bone marrow cell acquisition procedure

(A) Sterilize hind legs by soaking in 75% ethanol.

(B) Remove the muscle tissue from the hind legs.

(C) Wash the hind legs with cell culture medium.

(D) Rinse the bone marrow cavity with cell culture medium.

(E) Disperse the cell clumps with a pipette.

(F) Filter the tissue clumps with a cell strainer and collect bone marrow cells.

6. Remove the muscles and connective tissues with surgical scissors and forceps (Figure 2B), then wash twice with DMEM/F12 to remove 75% alcohol (Figure 2C).

7. Separate the femur and tibia, remove the epiphyses, and aspirate 1 mL of cell culture medium with an insulin syringe to rinse the bone marrow cavity and collect bone marrow cells (Figure 2D).

Note: Be careful with sharp objects.

8. Disperse the cell clumps with a pipette (Figure 2E) and filter out the tissue clumps with a 70 μm cell strainer (Figure 2F), then centrifuge at 500× g for 5 min and discard the supernatant.

9. Add 3 mL of red blood cell lysis buffer, mix thoroughly, and then lyse for 3 min at room temperature, immediately terminating with cell culture medium (containing 10% FBS).

10. Centrifuge at 500× g for 5 min. Discard the supernatant, resuspend it with cell culture medium, and counted.

11. Prepare a suspension of 106 cells per mL in medium containing M-CSF (50 ng/mL) and gentamicin (20 μg/mL). Add 10 mL of cell suspension per dish in a sterile 10 cm polystyrene cell culture-treated dish.

12. Incubate the dish at 37°C and 5% CO2 in a cell culture incubator.

Day 2.5

Timing: 30 min

13. Supplement with 5 mL of cell culture medium containing M-CSF (50 ng/mL) and continue incubation.

Day 3

Timing: 10 min

14. Measure the OD600 of BCG-EGFP cultures (Second passage, OD600–0.8).

15. Discard 3.5 mL of culture and supplement with 3.5 mL of fresh mycobacteria culture medium containing 50 μg/mL kanamycin (OD600–0.2).

16. Incubate in a shaker at 37°C, 100 rpm.

Day 5

Timing: 60 min

17. Discard the medium, wash the dish with 5 mL of PBS, and add 3 mL of trypsin-EDTA to detach the BMDM cells at 37°C for 5 min.

18. Add 3 mL of cell culture medium to terminate digestion, collect the digested cells with a pipette and centrifuge at 500× g for 5 min.

19. Discard the supernatant, resuspend the cells with cell culture medium and counted.

20. Prepare a suspension of 3×105 cells per mL in medium containing M-CSF (50 ng/mL). Add 300 μL of cell suspension per well into flat-bottomed 48-well cell culture plate (9 ×104 cells/well) and incubate overnight to let the BMDM cells adhere.

Note: The number and duration of passages for other BCG strains need to be adjusted according to their growth rate to ensure that the bacteria are in a logarithmic growth phase when used to prepare single bacterial suspensions.

Preparation of BCG single bacterial suspensions

Day 6

Timing: 60 min

21. Take 2 mL of logarithmic growth phase (OD600 ∼ 1) BCG culture and centrifuge at 2000× g for 1 min.

22. Discard the supernatant, resuspend with 1 mL PBST, and centrifuge at 2000× g for 1 min.

23. Discard the supernatant, gently resuspend with 1 mL PBST, and centrifuge at 500× g for 2 min.

Note: Resuspend gently and see the sediment gradually disappear.

24. Take the upper single bacterial suspension and measure the absorbance value at 600 nm to calculate the concentration of BCG in the suspension (Concentration = 1 × OD600 × 108 CFU/mL).

Induction of trained immunity in BMDM cells in vitro

25. Prepare a BCG suspension of 3 × 105 CFU per mL in medium containing M-CSF (50 ng/mL) (Figure 3A).Figure 3 Cytokine production by BCG-trained BMDM cells upon LPS restimulation

(A) Schematic diagram of trained immunity in BMDM cells.

(B) Cytokine profiles in control and BCG-trained BMDMs upon LPS restimulation. Unpaired t test was used to compare groups: ∗∗∗ P < 0.001. ∗∗∗∗ P < 0.0001. Values are expressed as mean ± SD.

26. Take the cell culture plate and discard the cell culture medium. Add 300 μL of BCG suspension per well into flat-bottomed 48-well cell culture plate and incubate at 37°C 5% CO2 for 24 h. The control group (Con) was added 300 μL of medium containing M-CSF (50 ng/mL).

Day 7

Timing: 30 min

27. Discard the cell culture medium, wash the cells twice with 1 mL PBS to remove extracellular BCG.

28. Add 300 μL cell culture medium containing M-CSF (50 ng/mL). Cells were cultured at 37°C, 5% CO2 in a cell culture incubator.

Day 9.5

Timing: 30 min

29. Discard the culture medium, add 300 μL fresh medium containing M-CSF (50 ng/mL). Cells were cultured at 37°C, 5% CO2.

Day 12

Timing: 30 min

30. Discard the culture medium, add 300 μL fresh cell culture medium containing 25 ng/mL LPS. Cells were cultured at 37°C for 24 h and collect the supernatant for cytokines analysis.

Day 13

Timing: 8 h

31. Detect cytokines in the supernatant according to the kit instructions (Figure 3B).

Note: BMDM and BCG single bacterial suspensions should not be stored for future use.

Expected outcomes

Herein, we describe the detailed protocol for determining BCG-induced trained immunity using murine BMDM cells. Firstly, the single bacterial suspension preparation protocol improves the reliability and reproductivity of BCG’s stimulation (Figure 1). Then, a standard murine BMDM differentiation method was used to obtain naïve macrophages derived from the bone marrow (Figure 2). As a result, high levels of inflammatory cytokines, which were regarded as surrogates of trained immunity, were detected in BCG-trained BMDM.

Limitations

This in vitro method of inducing trained immunity may not reflect the complexity of in vivo experiments. The protocol is optimized for mouse BMDM cells and may require further adaptation and optimization regarding scheduling concentrations for models with other cell types and donors.

Troubleshooting

Problem 1

The concentration of the harvested BCG single bacterial suspension is too low (e.g., OD600 < 0.2) (related to step 24).

Potential solution 1

Preparation of single bacterial suspensions requires very slight resuspension of the BCG precipitate to avoid large clumps. Alternatively, the concentration and volume of the initial BCG culture can be increased to obtain a higher concentration of a single bacterial suspension.

Problem 2

A large number of bone marrow cells die (related to step 11).

Potential solution 2

It could be that the 75% alcohol killed the cells. Do not break the femur and tibia in Steps 5–6 in Day 1 and wash well to remove the alcohol. A clear schematic of the surgical operation has been described in the published protocols.9,10

Problem 3

Not enough bone marrow cells were harvested. About 2 × 107 bone marrow cells can be harvested from a hind leg of one mouse (related to step 11).

Potential solution 3

Cut the femur and tibia keeping as much of the marrow cavity as possible. Or take more hind legs.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Xiao-Yong Fan (xyfan008@fudan.edu.cn).

Technical contact

Jinchuan Xu (2055046433@qq.com).

Materials availability

This protocol does not generate new unique reagents.

Data and code availability

This protocol does not generate or analyze any datasets or codes.

Acknowledgments

This work was supported by grants from the National Key Research and Development Program of China (2022YFC2302900 , 2021YFC2301503 ), National Natural Science Foundation of China (82171815 , 82171739 ), and Shanghai Municipal Health Bureau (2022XD060 ). The authors thank the Home for Researchers for drawing the graphical abstract.

Author contributions

Methodology, J.-C.X.; data analysis, J.-C.X.; writing, J.-C.X.; review and editing, Z.H., J.-C.X., and X.-Y.F.; supervision and funding, X.-Y.F. and Z.H.

Declaration of interests

The authors declare no competing interests
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