The overall goal of the following experiment is to rapidly generate a population of GR one positive enriched leukocytes containing viable myeloid derived suppressor cells or M DSCs from mouse spleens for use in in vivo and in vitro assays. First spleens from several mice are pooled and processed to generate a single cell suspension. To quantify the MDSC percentage prior to enrichment, a small aliquot of the pooled cells are transferred to a 96 well V bottom plate.
MAC one and a GR one surface antigens are labeled and the labeled cells are transferred to fax tubes for flow cytometric analysis. The remaining pooled leukocytes are stained with a fluorochrome conjugated antibody against GR one and incubated with magnetic microbeads, which bind to the fluorochrome on the anti GR one antibody. The GR one positive and GR one negative populations are then separated using an AutoMax pro separator to assess the success of GR one enrichment of the population.
A small aliquot is taken and analyzed for GR one and MAC one expression to evaluate MDSC percentages. Comparison of the resulting immunophenotyping data demonstrate an increase in MDSC percentages in the GR one enriched leukocyte population as compared to the non enriched population. In addition, there is an increase in naive MDSC yielding a population comparable to tumor bearing MDSC.
The main advantage of this technique over existing methods such as conventional flow staining and high speed flow cytometry based sorting of M DSCs from lymphoid tissues is that our master mix technique prevents significant variation in our results by ensuring equal surface staining of pool leukocytes, foren typing and AutoMax sorting of m DSCs and GR one positive cells respectively. Demonstrating the procedures will be Nadine Nelson, a graduate student from our laboratory, and Dr.Haw sra, director of the USF flow cytometry core facility. The following procedure should be performed in a sterile environment under a biological safety hood, cells and antibodies kept on ice.
Whenever possible, assemble the cell dissociation sieve by inserting the mesh screen into the opening of the cup toward the bottom. Then insert the retaining ring into the threaded area with the slotted side up and use the ring key to tighten the retaining ring to secure the screen. Place the assembled sieve in a Petri dish containing 10 milliliters of one XPBS pool.
Spleens taken from six to eight week old tumor bearing or naive control C 57 black six mice, and place them in the mesh screen of the cell dissociation sieve. Then use a glass pestle to grind the spleens through the mesh screen into the petri dish. Repeat this process for each treatment group of mice.
Next, using a five milliliter serological pipette, apply the cell suspension to a 70 micron cell strainer and allow it to filter into a 50 milliliter conical tube. Centrifuge at 250 to 300 times G for five minutes following the spin. Remove the supernatant and resuspend the pellet by adding five milliliters of RBC lysis, buffer per spleen and pipetting up and down vigorously incubate at room temperature for five minutes After the incubation, stop the reaction by adding 20 milliliters of PBS and pipetting up and down vigorously.
Then centrifuge at 250 to 300 times G for five minutes after removing the supernatant, the pellet in 20 milliliters of sterile PBS and count cells. Using trian blue and a hemo cytometer resus suspend the cells at a concentration of one times 10 to the seven to two times 10 to the seven cells per milliliter in 3%Staining medium. Achieving optimal staining is the most difficult aspect of this procedure.
This can be successfully carried out by titrating antibodies before. Use preparing master mixes and surface staining in small volumes as described in this protocol. To stain the cells for flow cytometric analysis, label the wells of a 96 well vbo plate for control and experimental samples and single stains.
For compensation controls, add five times 10 to the fifth to one times 10 to the sixth cells in 50 microliters to each of the labeled wells. Then centrifuge the plate at 250 to 300 times G for five minutes after centrifugation. View the plate from below.
Looking up to ensure that pellets are present in the wells, prepare a master mix of mouse BDFC block containing rat anti muse CD 1632 monoclonal antibody diluted in 3%staining medium in a 1.5 milliliter micro centrifuge tube on ice vortex. Then briefly centrifuge for five seconds holding the 96 well V bottom plate over a waste container or sink. Quickly invert the plate over and back to remove the supernatant without disrupting the cell pellets.
Then add 50 microliters of the FC block master mix to all of the pellets in the 96 well V bottom plate mixed by gently pipetting up and down. Place the plate on ice and incubate for 15 minutes in the dark centrifuge plate at 250 to 300 times G for five minutes. Prepare a master mix of Mac one fitzy and GR one A PC antibodies diluted in 3%Staining medium in a 1.5 milliliter micro centrifuge tube on ice.
Vortex briefly centrifuge for five seconds After the spin, invert the plate to remove supernatant from each well then add 50 microliters of the antibody mix to control or experimental pellets. Mix well by gently pipetting up and down for single stain compensations, add 50 microliters of diluted MAC one F GR one A PC or DPI in 3%staining medium to the appropriate wells. For the no stain control, add 50 microliters, 3%staining medium to the appropriate well and mix.
Then incubate the plate for 30 minutes in the dark on ice. During the incubation label fax tubes to correspond to each well of the plate and add 200 microliters of 3%staining medium to each fax tube. After the 30 minute incubation time is up, centrifuge the plate at 250 to 300 times G for five minutes.
After the spin, remove the supernatants as before. Then wash the pellets by adding 100 microliters of 3%staining medium to each pellet. Mix well by gently pipetting up and down.
Centrifuge the plate again for five minutes. Repeat, wash, step once more following the spin, remove the natin as before and resuspend each pellet in 100 microliters of 3%staining medium and mix well. Next, transfer 100 microliters of the resuspended cells from each well of the plate to the respectively labeled fax tube.
Then add 75 nanograms per milliliter DPI to the control and experimental samples and the DPI single stain compensation control. Acquire cytometric data acquisition to determine MDSC percentages as described in the accompanying document next to enrich GR one positive leukocytes aliquot one times 10 to the seventh of the remaining unstained leukocytes into appropriately labeled fax tubes and centrifuge them at 250 to 300 times G for five minutes. Add 50 microliters of a one to 10 dilution of GR one PE antibody in max buffer and incubate for 15 minutes at four degrees Celsius in the dark.
Following the incubation, add two milliliters of max buffer to each fax tube and centrifuge at 250 to 300 times G for five minutes. Following the spin, discard the supernatant. Then add 200 microliters of a one to four dilution of anti PE microbeads in max buffer to each tube and incubate for 15 minutes at four degrees celsius in the dark.
Add two milliliters of max buffer to fax tubes and centrifuge at 250 to 300 times G for five minutes. Then resuspend the pellet in three milliliters of staining buffer filter through a 70 micron strainer into a new labeled 50 milliliter conical tube. This protocol, we are using automatic magnetic separation.
Magnetic separation is a great choice of methods. If you need to separate a large number of cells fast cost effectively, and based on a surface marker, make sure that you check your results at the end of it with flow cytometry. To prepare and prime AutoMax pro separator, ensure that all bottles are filled with the appropriate solutions.
Turn the instrument on. Then after initialization, examine the status of fluid containers and columns. All symbols should be green.
Next on the menu, select separation from the upper menu bar followed by wash. Now from the lower menu bar, select rinse from the pop-up option followed by run to start the priming process. Once the priming process is successfully completed, the instrument will then display that it is ready for separation.
Under the status menu, place the 50 milliliter conical tube with magnetically labeled cells in row A of an appropriately sized chilled tube rack. Then place a 50 milliliter conical tube for negative fraction collection in row B and 15 milliliter conical tube for positive fraction collection in row C, choose POS cell S cell separation program for positive selection of labeled target cells in sensitive mode from samples. When the program begins, magnetically labeled target cells will be retained on the AutoMax column while unlabeled cells are released into the negative fraction collection tube in row B.The magnet will then be retracted and the labeled target cells will be released into the positive collection tube in row C of the tube rack.
After the enrichment, use trian blue and a hemo cytometer to recount GR one positive and GR one negative fractions. Then resuspend the cells at one times 10 of the seven cells per milliliter to two times 10 of the seven cells per milliliter in 3%staining medium and transfer 50 microliters to correspondingly labeled fax tubes. Stain cells with MAC one and DPI viability stain for flow cytometry and single stain compensations.
Perform flow cytometric data acquisition to determine GR one positive and GR one negative percentages and to also compare MDSC percentages pre and post AutoMax and enrichment Spleens were harvested from pancreatic tumor bearing and naive mice and processed into single cell suspensions as described in this video article flow cytometry was performed to examine naive leukocytes surface stained with MAC one and GR one fluorescent conjugated antibodies as shown here, only a very low percentage, 1.19 of naive MAC one and GR one positive. MDCs is seen prior to AutoMax enrichment. However, following enrichment of the GR one positive population, this cell subpopulation is significantly larger, 10.5%from naive and is now comparable to 25.3%from tumor bearing mice.
Following this procedure, other methods including accelerated MDSC, flow cytometry based sorting in vivo adopted transfer experiments, quantitative real-time PCR and western blotting can be performed to answer additional questions regarding the molecular mechanisms responsible for MDSC expansion, migration differentiation, and their suppression of immune responses.
