The overall goal of these procedures is to quickly isolate highly purified neutrophils from human blood samples by density gradient centrifugation to investigate their role in lymphoma cell sensitivity to cancer therapy using two D and three D cell culture models. These methods can help answer key questions about the interactions between the innate immune system and cancer such as what role does a neutrophil mediated tumor cell responses play in cancer therapy. The main advantage of density gradient separation is the ability to isolate pure immune cell populations in a short period of time without exposing the cells to function altering stimuli.
To isolate the primary leukemic cells and neutrophils, begin by adding 15 milliliters of EDTA treated blood from chronic lymphocytic leukemia patient samples into individual, sterile 50 milliliter tubes. Dilute the blood with 15 milliliters of RPMI. Then carefully and slowly add 15 milliliters of room temperature density gradient solution under the blood, taking care not to mix the layers.
After separating the cells by centrifugation, four distinct phases will be visible. The platelets and plasma at the top, a white ring of mononuclear cells, the density gradient solution, and the granulocytes and erythrocytes at the bottom. Using a plastic pasture pipette, first transfer the white ring of primary leukemic cells in a new 50 milliliter tube.
Wash the cells with up to 50 milliliters of PBS with calcium and magnesium. And re-suspend the palate in five milliliters of fresh PBS. Then, add 45 more milliliters of PBS to the cells, and spin down the cells again.
After the second wash, re-suspend the palate in complete RPMI medium. And count the number of viable primary leukemic cells. Next, remove the platelet plasma and density gradient layers, and add 25 milliliter of PBS and 25 milliliters of Dextrin in sodium chloride to the granulocyte erythrocyte phase.
Mix the tubes ten times by inversion. Then, let the cells settle for 30 minutes at room temperature. At the end of the equilibration, transfer the upper red blood cell poor neutrophil layer into a sterile, 50 milliliter tube and spin down the cells.
Resuspend the palate in five milliliters of PBS and lyse the red blood cells with 45 milliliters of red cell lysis buffer. After 15 minutes in the dark at room temperature, spin down the cells again and wash the palate in 50 milliliters of PBS. At the end of the wash, re-suspend the cells in the complete RPMI medium and count the number of viable neutrophils.
Dilute both groups of cells to a 300, 000 cell per 50 microliters of RPMI concentration and divide the samples between the appropriate number of five milliliter fax tubes. After centrifugation, resuspend the pellets in 50 microliters of PBS supplemented with FBS. To determine the purity of the isolated cell subsets, label the aliquant of primary leukemic cells with anti humanCD19 antibody conjugated to APC and the aliquant of the purified neutrophils with a mixture of anti human antibodies as listed in the table for 30 minutes in the dark at four degrees Celsius.
At the end of the incubation, wash the cells in 500 microliters of FBS supplemented PBS and resuspend the palates in 200 microliters of fresh FBS PBS. Then, analyze the purity of the cell populations by flow cytometry, taking care to color compensate the samples. To co-culture the primary leukemic cells with the autologist neutrophils, see 200, 000 cells per milliliter of leukemic cells alone or with autologist neutrophils in a 24 well plate in complete RPMI medium.
Mix the cells carefully. Then treat the cells with Brutton's Tyrosine Kinase Inhibitor for 24 hours at 37 degrees Celsius and five percent CO2. The next day, collect an aliquant of the cells in a five milliliter plastic fax tube.
Then, spin down the cells and wash the pellets in one milliliter of FBS PBS. After the second centrifugation, label the cells aninexine five and propidium iodide according to the manufacturer's instructions for ten minutes at room temperature in the dark. Then, analyze the neutrophils and primary leukemic cells by flow cytometry using the illustrated gating strategy.
To set up a three D co-culture, add 50, 000 RL lymphoma b-cells alone or mix with HL60 differentiated cells in sterile 15 milliliter tubes. Spin down the samples and resuspend the pellets in 300 microliters of basement membrane matrix, using a one millimeter pipette tip with the opening cut to two to three millimeters. Next, incubate 300 microliters of the cells in each well of a 24 well plate for 30 minutes at 37 degrees Celsius and five percent CO2.
At the end of the incubation, add one milliliter of complete RPMI medium to each well and return the threeD cultures to the incubator for another seven days with medium changes every two days. On day five, add ten nanomolar of incristine to the cultures. After one week of culture, aspirate the medium and wash each well two times with one milliliter of ice cold PBS.
Then, add three milliliters of ice cold PBS with EDTA to each well and scrape the bottom of the wells with a 200 microliter pipette tip to detach the gels. Shake the plate gently on ice for 30 minutes. Then, transfer the cell suspensions to individual sterile 15 milliliter tubes for gentle shaking on ice for another 30 minutes.
Confirm the appearance of a homogenous cell suspension. Then, spin down the cells followed by a PBS wash and resuspend the pellets in fresh PBS with FBS. Finally, label the cells with the appropriate antibodies and viability dyes as just demonstrated and analyze the RL and HL60 differentiated cells by flow cytometry using the illustrated gating strategy.
Analyzing all the events of the isolated cell populations by flow cytometry demonstrates that this separation method results in a highly pure single peak CD19 expressing primary leukemic cell population. And a greater than 90 percent pure neutrophil population positive for the leukocyte common antigens CD45, and the CD15 and CD16 antigens expressed by mature neutrophils. Gaiting on the primary leukemic cells from neutrophil co cultures demonstrates that the neutrophils exert a protective effect on cell survival against the tyrosine inhibitor ibrutinib with a significantly higher number of viable primary leukemic cells compared to the culture of primary leukemic cells alone.
Differentiated HL60 cells are more stable in three D cultures than neutrophils. After induction of their granulocytic development, these cells are smaller than undifferentiated HL60 cells and express higher levels of CD11B and CD38. Differentiated HL60 cells also exhibit multi lobed nuclei.
These cells exhibit a protective effect on the RL cells with a significantly higher number of viable RL cells in three D co cultures after vincrostin treatment compared to vincrostin treated lymphoma cells cultured alone. Once mastered, the density gradient centrifugation method can be completed in 90 minutes. After their development, these techniques paved the way for researchers in the field of cancer biology to explore the impact of various white blood cells on tumor malignancy in preclinical models and patient samples.
After watching this video, you should have a good understanding of how to quickly and reliably isolate mononuclear cells and the neutrophils, analyze the effect of neutrophils on mediating tumor cell responses to anti cancer agents, and set up a three D co culture.
