FJGIL

Mice: C57BL/6 (B6) and BALB/c (Balbc)

Animal models, especially mice, are the most common way to study the effects of medicine thoroughly before attempting human clinical trials. Commonly used mice for immunological studies are the C57BL/6 (abbreviated B6) and BALB/c (abbreviated Balbc) mouse strains.

C57BL/6 Mouse Strain BMDC in Immunology

The C57BL/6 mouse strain is a popular choice for immunology research because it is a well-characterized strain with a high degree of genetic homogeneity. BMDCs, or bone marrow-derived dendritic cells, are a type of immune cell that plays a key role in the initiation of adaptive immunity. They are derived from hematopoietic stem cells in the bone marrow and mature in the spleen and lymph nodes. BMDCs are able to present antigens to T cells, which helps to initiate the immune response.

C57BL/6 BMDCs have been used to study a variety of immunological processes, including:

T cell activation: C57BL/6 BMDCs can be used to activate T cells in vitro. This allows researchers to study the molecular mechanisms involved in T-cell activation.
Antigen presentation: C57BL/6 BMDCs can be used to present antigens to T cells. This allows researchers to study the role of BMDCs in the presentation of antigens to T cells.
Immune tolerance: C57BL/6 BMDCs can be used to induce immune tolerance. This allows researchers to study the mechanisms involved in immune tolerance.

C57BL/6 BMDCs are a valuable tool for immunology research. They can be used to study a variety of immunological processes, including T-cell activation, antigen presentation, and immune tolerance.

Here are some additional benefits of using C57BL/6 BMDCs for immunology purposes:

They are easy to obtain and culture.
They are relatively inexpensive.
They have a well-defined genetic background.
They have been used in a wide variety of immunological studies.

Sure, here is a blog post about the uses of the BALB/c mouse strain BMDC for immunology purposes:

BALB/c Mouse Strain BMDC in Immunology

The BALB/c mouse strain is also a popular choice for immunology research because it is also a well-characterized strain with a high degree of genetic homogeneity.

BALB/c BMDCs have been used to study a variety of immunological processes, including T cell activation, antigen presentation, and immune tolerance as described above.

Here are some of the key differences between C57BL/6 and BALB/c BMDCs:

C57BL/6 BMDCs are more efficient at presenting antigens to T cells than BALB/c BMDCs.
C57BL/6 BMDCs are more potent at inducing immune tolerance than BALB/c BMDCs.
C57BL/6 BMDCs are more resistant to apoptosis than BALB/c BMDCs.

These differences in BMDCs from the two strains can be attributed to their different genetic backgrounds. C57BL/6 mice are known to have a higher expression of genes involved in the immune response, while BALB/c mice have a higher expression of genes involved in the regulation of the immune response.

The choice of which BMDC strain to use for immunology research will depend on the specific research question being asked. If the research question is focused on the immune response, then C57BL/6 BMDCs may be a better choice. If the research question is focused on the regulation of the immune response, then BALB/c BMDCs may be a better choice.

BALB/c Mice as a Model for Immuno-Stimulating Drug Compounds

Benefits of Using BALB/c Mice:

There are several benefits to using BALB/c mice for immunology research:

Susceptibility to immunomodulation: BALB/c mice are more susceptible to immunomodulation than other strains of mice. This means that they are more likely to develop an immune response when exposed to immuno-stimulating drug compounds. This makes them a good choice for studies that are designed to investigate the mechanism of action of these compounds.

Key Differences Between BALB/c and Other Strains of Mice:

Here are some of the key differences between BALB/c and other strains of mice that make them more susceptible to immunomodulation:

Lower levels of regulatory T cells: BALB/c mice have lower levels of regulatory T cells than other strains of mice. Regulatory T cells are a type of immune cell that helps to suppress the immune response. Their lower levels in BALB/c mice make them more susceptible to immunomodulation.
Higher levels of pro-inflammatory cytokines: BALB/c mice have higher levels of pro-inflammatory cytokines than other strains of mice. Pro-inflammatory cytokines are proteins that promote inflammation. Their higher levels in BALB/c mice also make them more susceptible to immunomodulation.

Th1 and Th2 Immune Response

To understand the Th1 and Th2 responses, we must first understand the basis of these immune responses. Cytokines are the hormonal messengers responsible for most of the biological effects in the immune system. These messengers can be functionally divided into two groups: those that are pro-inflammatory and those that are anti-inflammatory but promote allergic responses.

T lymphocytes are a major source of cytokines, and the main two subsets can be distinguished by their surface molecules known as CD4 and CD8. CD4 T lymphocytes, also known as helper T cells, are the most prolific cytokine producers. They produce both Th1-type cytokines and Th2-type cytokines.

Th1-type cytokines, such as interferon gamma (INFg), tend to produce pro-inflammatory responses which perpetuate autoimmune responses and kill intracellular pathogens. Excessive pro-inflammatory responses can lead to uncontrolled tissue damage, so the Th2 response is the mechanism to counteract this. In excess, the Th2 response will counteract the Th1-mediated pro-inflammatory action. The Th-2 type cytokines associated with this counteraction include interleukins 4, 5, and 13, which are associated with the promotion of allergic responses, and interleukin 10, which has more of an anti-inflammatory response. Optimally, a human would produce a well-balanced Th1 and Th2 response suited to the immune challenge.

In tumor studies, TLR4 has been reported to enhance the function of antigen-presenting cells (APCs), increase the production of pro-inflammatory cytokines (including IFNs), and boost the cytotoxic responses of both cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. These immune-stimulating mechanisms can be initiated by TLR4 agonists in cancer treatment. In this lab, the efficacy of synthetic TLR4 agonists in combination with mRNA vaccines is tested as an immune-stimulating agent.

Making Bone Marrow-Derived Dendritic Cell (BMDC) Culture

Bone Marrow-Derived Dendritic Cells (BMDC) are potent antigen-presenting cells (APC) that have the unique capacity to initiate primary immune responses. BMDC is used in clinical studies to induce immunity against infectious diseases and malignant cells. This BMDC immunostimulatory capacity makes them attractive candidates for modulating immunity against pathogens, including viruses and bacteria, and also against malignant cells. In mice, BMDC can be generated in vitro from bone marrow-derived progenitor cells (from the femur, tibia, and knee) in the presence of the cytokine Granulocyte Monocyte Colony Stimulating Factor (GM-CSF). These cells are primarily used to study the induction of anti-tumor immunity in mouse model systems in vivo.

Harvest leg bones from mouse

1. Spray the mouse thoroughly with ethanol and pin down the stretched-out mouse by all 4 paws

3. Lift the skin on the abdomen and snip it open while leaving the peritoneum intact

4. Cut and pull back the skin covering the lower abdomen and pin it down

5. Use tweezers to lift the skin on each hind leg and then use scissors to cut near the torso and then slice toward the paw as far as possible

Pull the skin away from the bone (both above and below) and pin down
No fur should be showing around the hind legs

6. Close scissors and use them to locate the femur

7. Slice away muscle near the femur by opening scissors parallel to the bone, both above and below the bone

8. Use tweezers to lift bone slightly

9. Turn the scissors so that the middle of the scissors is in position to cut the femur and push toward the torso as far as possible

10. Continue lifting the bone (not too high) and cut the femur

11. Using tweezers to lift the bone, turn the scissors and cut the thighbone at the knee, and set it aside in a small Petri dish

12. Use tweezers to lift the tibia

13. Use scissors to cut all the muscle and tendons that impedes lifting the bone

14. Continue to hold the tibia with tweezers, and use the middle of scissors to cut the bone at the ankle

15. Cut the knee away from the tibia, and set bones aside in a small petri dish

Flush bone marrow and plate cells

1. Put 30 ml of RP10 in a 50 ml centrifuge tube

2. Use a 10 ml syringe to take up RP10

3. Attach a 27G (0.5 in.) needle to the syringe

4. Use the needle to flush marrow from recovered bones into the centrifuge tube

Also wash the outside of the bone if necessary, particularly if the bone has been broken in recovery

5. Pipet up and down to break chunks and make a single-cell suspension as much as possible

6. Centrifuge cells (in RP10) at 300 g (1100 rpm) for 5 min

7. Re-suspend the cell pellet in 20 ml of RP10 and filter through a 100 um cell strainer, making sure to break any chunks in the filter

8. Stain cells with Turk’s stain, count with a hemocytometer, and adjust concentration to ~6e6 cells/ml

Calculation: cell concentration desired (cells/ml) / cell concentration (cells/ml) = Ratio
Ratio * Volume desired (ml) = Volume cells (ml)
Volume desired (ml) – Volume cells to use (ml) = Volume media to add (ml)
Volume media (ml) + Volume cells (ml) = Volume desired (ml) with [cell] desired (cells/ml)

9. Plate 5 ml each of 30e6 cells/dish for B6 mice (or 24e6 cells/dish for Balbc mice) in 145 x 20 mm Petri dishes

10. Add 15 ml of RP10 to each dish

11. For 4 dishes, add 20 ul of mGM-CSF (0.1 mg/ml) to 20 ml of RP10

12. Split the RP10 with mGM-CSF between 4 Petri dishes (5 ml each), bringing the final volume to 25 ml per dish

Feed cells (3 days after plating)

1. Add 20 ml RP10 to each of the 4 dishes, bringing the volume to 45 ml

2. For 4 dishes, add 20 ul of GM-CSF (0.1 mg/ml) to 20 ml of RP10 in a 50 ml tube

3. Add 5 ml of RP10 with GM-CSF to each dish, bringing the final volume to 50 ml per dish

Feed cells (6 days after plating)

1. Label 50 ml centrifuge tubes with the corresponding label on each Petri dish

2. From each of the plates, take out 25 ml of the cell suspension and spin down the cells in their corresponding centrifuge tubes at 300 g for 5 minutes

3. Trash the supernatant (old medium) and add 20 ml of RP10 to each of the 4 tubes to resuspend the cells and add back to the plate where the cells came from

4. For 4 dishes, add 20 ul of GM-CSF (0.1 mg/ml) to 20 ml of RP10 in a 50 ml tube

5. Add 5 ml of RP10 with GM-CSF to each dish, bringing the final volume to 50 ml per dish

Harvest BMDC (8 days after plating)

1. Set aside one 50 ml centrifuge tube for each petri dish of cells

2. Transfer 25 ml of medium plus cells from the dish to the centrifuge tube

3. Gently pipet the remaining medium up and down 2~3 times to harvest the remaining cells and transfer them to the centrifuge tube

* Careful, excessive pipetting can stimulate the cells

4. Centrifuge tubes with cells from all dishes at 300 g for 5 min

5. Resuspend cells in RP10, combining cells from all tubes in one tube

* Recommended volume: 20 ml

6. Count cells (stain with Trypan Blue, count with CountessII)

* The number should be around 8e6 cells/ml. The viability of the cells should be above 90%. If lower than these numbers, please check with your supervisor

7. Dilute cells to your desired concentration for plating in 96-well plates

* For cytokine release, plate 10e5 cells/well. For FACS, plate 2e5 cells/well in non-treated plates

8. Let cells rest for 2 hrs before treatment at 37°C

These cells are very sensitive. Use minimal pipetting throughout this entire protocol in order to be as gentle as possible.