The overall goal of this experiment is to genetically modify the murine myeloid 32D-G-CSF-R cells by over expressing or silencing the gene of interest and determining the effect of these changes on proliferation and neutrophilic differentiation of the cell line. This method can help to answer key questions in the field of hematology such as how your protein of interest can be involved in growth and differentiation of myeloid precursor cells. The main advantage of this cell line is that it possesses unlimited proliferation capacity, is susceptible to genetic manipulations, the cost is relatively low, and allows a degree of biological simplification required in certain experimental approaches.
Demonstrating the procedure today will be Petr Danek, a PhD student in my laboratory. Prepare 250 milliliters of RPMI-1640 Medium supplemented with heat-inactivated 10%fetal bovine serum and 10 nanograms per milliliter of murine interleukin-3. Then prepare 50 milliliters of differentiation medium.
In a 16 centimeter Petri dish, seed single cell suspension of Bosc23 cells in 18 milliliters of DMEM medium supplemented with 10%fetal bovine serum. Culture for about 24 hours until the confluency reaches 80%Then combine MSCV retroviral construct, PCL echo packaging vector, polyethylenimine, and reduce the serum medium without antibiotics. Incubate this mixture in a Flowbox at room temperature for 20 minutes.
Then carefully replace the Bosc23 cell culture medium with 16 milliliters of pre-warmed DMEM medium supplemented with 2%fetal bovine serum maintained at 37 degrees Celsius. Next, add the retroviral mixture drop wise on the Bosc23 cells. Then incubate the culture at 37 degrees Celsius for four hours.
Once the incubation is over, aspirate the Bosc23 culture medium and add 18 milliliters of pre-warmed DMEM medium with 10%fetal bovine serum. Incubate the cells at 37 degrees Celsius for 48 hours. Use a 25 milliliter serological pipette to draw the Bosc23 virus-containing medium into a 50 milliliter conical tube.
Store the tube at four degrees Celsius. Then add 18 milliliters of pre-warmed DMEM medium with 10%fetal bovine serum to the Bosc23 cells and culture the cells for 24 hours. Centrifuge the viral supernatant at 1, 500 times g for 10 minutes at four degrees Celsius.
Then aliquot the virus and snap freeze it with liquid nitrogen to store at minus 80 degrees Celsius. In each well of a six-well plate, seed NIH/3T3 cells in three milliliters of DMEM medium supplemented with 10%fetal bovine serum to titer one virus. Culture the cells overnight at 37 degrees Celsius.
The following day, dilute the virus with pre-warmed medium without vortexing the viral particles. Aspirate the culture medium from the NIH/3T3 cells and add one milliliter of diluted virus in each well. Incubate at 37 degrees Celsius overnight.
The next day, carefully aspirate the diluted viral medium and replace with two milliliters of DMEM medium with 10%fetal bovine serum and again incubate at 37 degrees Celsius overnight. After the incubation, aspirate the old media and replace with two micrograms per milliliter puromycin-containing DMEM medium. Continue culturing the cells every three days or unless the media turns yellow and replenish with fresh media containing puromycin.
Between the 10th and 12th day post infection, aspirate the media from the wells and gently wash with phosphate buffered saline. Then stain with one milliliter of crystal violet solution at room temperature for two minutes. After staining, carefully wash with phosphate buffered saline twice.
Count the total number of blue colonies present in each well under a microscope. Seed the 32D-G-CSF-R cells in a six-well plate. Add virus at a multiplicity of infection between 10 and 40.
Then add polybrene to a final concentration of eight micrograms per milliliter to the cells and incubate at 37 degrees Celsius for six hours. After incubation, pool the cells into a 15 milliliter tube and centrifuge at 450 times g for five minutes at four degrees Celsius. The 32D cells may partially lose their ability to differentiate if grown to the concentrations higher than one million cells per ml.
Therefore, it's very important to split these cells on time and do not overgrow the cultures. Discard the supernatant and resuspend the cells in a T25 culture flask containing six milliliters of RPMI-1640 Medium supplemented with heat-inactivated 10%fetal bovine serum and 10 nanograms per milliliter of murine interleukin-3. Incubate the cells at 37 degrees Celsius for 48 hours.
Wash the 32D-G-CSF-R cells twice with RPMI-1640 Medium without cytokines. Then seed the cells in one milliliter of differentiation medium in a 24-well plate at day zero and incubate at 37 degrees Celsius overnight. The cells will take seven to nine days to differentiate into mature granulocytes.
Then count the number of cells using trypan blue to exclude dead cells. Since IL-3 may inhibit the differentiation process, it is absolutely critical to remove all traces of the cytokine before culturing the cells in GCSF. Cytospin the cells on a glass slide in a centrifuge machine with necessary adapters at 20 times g for five minutes.
Then fix the cells in methanol for five minutes at room temperature and allow the cells to dry. Stain the cells using May-Grunwald Giemsa staining following the manufacturer's protocol. Once stained, visualize the cells that have taken the stain using 40X magnification.
Based on cellular morphology, quantify the number of blasts, intermediately differentiated cells, and mature granulocytes in the stained cells. Shown here is a graphical representation demonstrating proliferation of 32D-G-CSF-R cells in interleukin-3 and granulocyte colony stimulating factor containing media. The x-axis represents the days of treatment.
The y-axis represents the cell count in logarithmic scale. The black plot shows proliferation when the cells are cultured in interleukin-3 containing medium. The blue plot shows declined cell proliferation when cultured in granulocyte colony stimulating factor containing medium.
Shown here are representative pie charts and May-Grunwald Giemsa staining showing the proportion and differentiation morphology of 32D-G-CSF-R cells in granulocyte colony stimulating factor containing medium. The pie charts demonstrate the proportion of blasts, intermediately differentiated cells, and neutrophils on granulocyte colony stimulating factor treatment at zero, two, four, and six days. Giemsa stained cells at day zero have large characteristic nuclei and dark cytoplasm.
On day six, the nuclear size is reduced and the cytoplasm is enlarged but not dark. Shown here are representative pie charts showing the higher proportion of blast cells when transduced with BCR-ABL fusion proteins compared to control MSCV vector in granulocyte colony stimulating factor containing media. Giemsa staining in the lower panel is representative of the cells when transduced with control or BCR-ABL fusion proteins.
The cells underwent neutrophilic differentiation at day six when transduced with MSCV control vector, but BCR-ABL transduced cells showed mostly blast cells and no mature neutrophils. Once mastered, this technique can be done in two days if it is performed properly. While attempting this procedure, it is important to remember to culture the 32D-G-CSF receptor cells at indicated concentrations.
Following this procedure, other methods like migration assays and survival experiments can be performed in order to answer additional questions. After watching this video, you should have a good understanding of how to handle the 32D-G-CSF receptor cell line covering genetic manipulation by lentiviral and retroviral transduction, expansion, differentiation, and assessment of proliferation and differentiation of these cells. Don't forget that working with viruses can be dangerous and precautions such as wearing gloves and protective glass should always be taken while performing this procedure.
