This procedure assesses in a semi-solid medium, the ability of hematopoietic progenitors to proliferate and differentiate along different hematopoietic lineages to begin culture. Freshly thawed CD 34 positive cells in the presence of cytokines to promote activation after two days. Perform retroviral transduction of a GFP expressing test construct by adding the activated CD 34 positive cells to a plate preloaded with virus.
48 hours later isolate the GFP positive cells by fax, then plate these cells in semi-solid methylcellulose medium supplemented with growth factors incubate for approximately two weeks until colonies appear on the surface. Finally, harvest the colonies, immobilize the cells on slides using cytosine and stain with right giza for microscopic determination of hematopoietic lineage and maturation stage. This method can provide mechanistic insight in studies of leukemogenesis, namely the effects of oncogenes on hematopoietic differentiation.
In order to see a culture quickly thaw a vial of frozen CD 34 positive cells at 37 degrees Celsius by gently shaking until a last small ice crystal is left and transfer the cell suspension to a 50 milliliter conical tube. Gently rinse off the remaining cells from the vial with one milliliter of room temperature, 2%F-B-S-I-M-D-M media and add it dropwise to the 50 milliliter tube while swirling gently. Now wait for three minutes.
Continue by slowly adding two milliliters of media while mixing gently and again equilibrate for three minutes. Repeat this procedure of adding media of equal volume to the diluted cell suspension at three minute intervals swirling gently in between additions until the final volume reaches 32 milliliters to collect the cells centrifuge at 250 G for 10 minutes at room temperature and remove the supernat leaving behind around 0.5 milliliter of media above the pellet. Proceed to wash the cells once by suspending the pellet in 20 milliliters of media and centrifuging at 200 G for eight minutes At room temperature, remove the sane and suspend the cells in complete IMDM medium at approximately half a million cells per milliliter.
Finally, to determine cell concentration, take 10 microliters of the suspension into a micro tube mixed with the same volume of 0.4%trian blue solution and count. Using a hemo cytometer dilute the cells to 10 to the fifth cells per milliliter by adding complete IMDM medium supplemented with the mix of activating cytokines culture, the cells in a humidified 37 degree Celsius incubator with 5%carbon dioxide for two days on the day of transduction count cells before starting virus preloading to determine the number of wells to be prepared in order to preco A 24 well non-treated tissue culture plate. Add 400 microliters of 25 micrograms per milliliter, retro actin solution to each well and incubate for two hours at room temperature in the laminar flow biosafety hood.
Remove the retro actin solution and code each well with 2%BSA by incubating for 30 minutes at room temperature. Meanwhile, thaw out the virus stock solutions and store on ice. Then aspirate the BSA solution load virus by adding 0.5 milliliter of virus preparation to each well and centrifuge at 2, 200 G at four degrees Celsius for 15 minutes.
Remove the virus solution from the wells and repeat the virus loading three more times. Finally, rinse each well with cold IMDM medium. Now collect the pre activated CD 34 positive cells by centrifugation and resuspend the cells in fresh complete IMDM medium supplemented with cytokines aliquot 0.15 million CD 34 positive cells into each of the virus coded wells, wells and incubate in a humidified 37 degree Celsius incubator with 5%carbon dioxide for two days in order to harvest the transduced cells gently but thoroughly.
Suspend the cells from the virus floated plates and filter the suspension through a 50 micron cell filter into a conical tube. Take 10 microliters of the cell suspension into a micro tube. Mix with an equal volume of trian blue solution and count cells using a hemo cytometer.
Rinse each well with 0.8 milliliter of cold HBSS with 0.02%EDTA and add to the same collection tube through a 50 micron CEL Trix cell filter. Pellet the cells by centrifugation and wash once with cold HBSS. Re suspend the final cell pellet in HBSS with 1%BA and keep the cells on ice in the dark for subsequent cell sorting, which is typically performed at a high speed cell sorter core facility based on experimental design.
Thaw the required number of aliquots of CFC assay media vortex vigorously to mix and let the tube stand for at least five minutes to let bubbles rise to the surface before adding cells. Now take 3000 virus transduced sorted cells into a sterile micro tube containing cold 2%F-B-S-I-M-D-M media and adjust the final suspension volume to 0.3 milliliters. Suspend the cells and transfer the entire cell suspension to a three milliliter aliquot of methyl cult growth Medium.
Create a homogenous cell suspension by vortexing vigorously. Let the tube stand still for three minutes. In order to plate the cells, attach a 16 gauge blunt end needle to a three milliliter syringe and draw of 2.2 milliliters.
Taking care to avoid uptake of large bubbles, push out 1.1, milliliter each into two 30 millimeter non-treated tissue culture dishes and spread out the mixture evenly by rotating place duplicate plates in a 100 millimeter plate together with a water dish. Continuing three liters of sterile water culture for two weeks. Characterize and score the colonies according to their morphology with an inverted microscope at 40 x magnification in a culture dish marked with a scoring grid.
In order to document the culture results, scan the entire CFC assay plate using a regular scanner at 600 DPI and take low power photo micrographs of representative colonies using an inverted microscope equipped with a color camera for further analysis of differentiation and proliferation cells from the entire CFC assay plate are recovered by suspending in several volumes of room temperature 2%F-B-S-I-M-D-M washed and counted for morphological analyses. Transfer 30, 000 cells that were recovered from CFC assay plates to a slide. Using a STOs spin centrifuge, air dry the slides overnight to efficiently stain the slides.
Set up an assembly line of nine staining vessels containing solutions in the following order. Absolute methanol right gemsa stock solution right gemsa buffer, 50%methanol and water, two vessels of water phosphate buffer pH six and finally two vessels of water. Place the slides into a slide carrier and dip in absolute methanol for two minutes and blood off excess methanol.
Immediately dip the slide carrier and write gemsa stock solution for five minutes. Transfer the carrier into right gemsa buffer pH 6.4 and incubate for 10 minutes. Dip the carrier twice in 50%methanol 10 times in water and another 10 times in the next water vessel and then five times in phosphate buffer.
pH 6.0. Transfer the carrier into water and incubate for two minutes. Repeat the wash in the last water vessel.
Now allow the slides to air dry completely in the carrier. Remove each slide and wipe the back of the slide with methanol soaked Kim wipes to remove stains. Place a drop of cyto seal 60 and a cover glass to seal.
Perform a 500 cell differential count for each GM sustained slide using a microscope. For the purposes of this experiment, cells are divided into five categories, primitive cells, intermediate myeloid cells, mature myeloid cells, intermediate erythroid cells, and mature erythroid cells. For example, when compared to control expression of the new 98 hawks, a nine oncogene increased the formation of red erythroid colonies.
This market impact of the oncogene is clearly evident when colonies are observed under low magnification. Further morphological examination by GEM sustaining provides information on the lineage and degree of maturation of the cell population. In this example, introduction of new 98 Hawks, a nine caused an overall increase in the numbers of cells with erythroid hyperplasia and inhibit inhibition of both erythroid and myeloid maturation.
This SA provides insight into hematopoietic cell differentiation by measuring the ability of an input cell preparation to proliferate, differentiate and form colonies in a semi-solid medium.
