The overall goal of this procedure is to generate an array of up to 252 distinctly labeled fluorescent target cells for immune assays using multi-parameter flow cytometry. This is accomplished by first labeling target lymphocytes with unique combinations and intensities of vital dyes such as CFSE and CTV. The next step is to pulse each distinct target lymphocyte population with candidate MHC Class one and MHC class two binding peptide epitopes.
This is followed by extensively washing the cells to maintain the specificity of the epitopes and then pooling the cells and injecting them into an animal. The final steps are to harvest the fluorescent tag array cells from specific tissues, label them and assess for MHC peptide specific responses. Ultimately, flow cytometry results can show CTL responses by measuring target cell death and they can show CD four positive T helper responses by measuring B cell target upregulation of activation markers On the major advantage of this technique over conventional in vivo CTL assays is that it allows a simultaneous measurement of over 250 target cells in one animal demonstrating the procedure will be Ben Quire, a research fellow in my laboratory.
The procedure requires lymphocytes collected from the isolated spleen and or lymph nodes of mice filtered and resuspended at up to 200 million cells per milliliter in 11.5 milliliters of RPMI containing 5%fetal calf serum. Preparing the FDA begins with labeling. 42 10 milliliter conical bottomed plastic tubes from one to 42 in all cells will be pulsed with seven different peptide epitopes at six different concentrations repeated six times to generate 252 discernible cell clusters.
Now, thoroughly resus suspend the lymphocytes by inverting the tube several times. Then add 1.9 milliliters of cell suspension to the 10 milliliter tubes labeled 37 through 42. Taking care not to wet the top half of the tubes.
Now uncapped tube 38 and lay it horizontally to the top of tube 38. Add 83 microliters of PBS. Then add 17 microliters of the appropriate CTV stock as presented in table two of the text protocol.
Once the tube is loaded, vortex it. Repeat this with tubes 39 to 42. Ensuring each tube gets a different CTV stock as listed in table two.
Now incubate the cells for at least five minutes at room temperature. Then add five milliliters of RPMI containing 5%FCS to each tube Thoroughly resuspend the cells by vortexing from each tube. Transfer one milliliter of suspension to six other tubes following the suggested loading pattern outlined in the text protocol during every transfer, it remains important to not wet the upper part of the loaded tube to label the cells with CFSE, remove the tube caps of tubes 31 to 36 containing one milliliter of cells and lay the tubes horizontally next to each of the tubes.
First, add 103 microliters of PBS, ejecting it onto the dry upper wall. Then add seven microliters of the appropriate CFSE stock according to table three of the text protocol and immediately vortex the tube. Repeat this with the tubes as grouped in table three.
Ensuring each tube gets a different CFSE stock. Incubate all the tubes for at least five minutes at room temperature. After waiting five minutes, wash the cells to finish the labeling.
Dilute each suspension with nine milliliters of room temperature RPMI with 5%FCS. Pellet the cells at 300 GS for 10 minutes at room temperature and remove the supernatants by aspiration using a transfer pipette the preparation of MHC Class one and MHC class two binding peptides is presented in the text protocol. This section shows how to pulse label the cells with these peptides.
First, resuspend the cell pellet in residual medium, for example, by running the tubes along a grill. Then bring the total volume of the cell suspension to 250 microliters in our PMI containing 5%FCS. Each cell pellet should need about 200 microliters of medium.
Next, add 250 microliters of pre-prepared peptide stocks listed in table five to the designated tubes. Follow the pattern presented in table one and be sure to include a control tube of PBS alone for calculating T cell responses. It is critical that each peptide epitope and each peptide concentration is assigned to a single tube and that this is recorded clearly based on the expected fluorescence of the cells in this tube.
Since the fluorescent signature of this cluster will define this peptide, mix each cell suspension by vortexing and then incubate the cells at 37 degrees Celsius for one hour. After the incubation, proceed with washing the cells to wash the cells. First, add five milliliters of ice cold RPMI with 5%FCS to each cell suspension and resuspend the cells by inverting the tubes.
Then carefully underlay each cell suspension with three milliliters of ice cold FCS sediment, the cells with 10 minutes of spinning at 300 G kept at four degrees Celsius. Use slow acceleration and braking to ensure the solution interface is maintained in each tube. Carefully aspirate off the RPMI then aspirate off the FCS leaving the washed cell pellet undisturbed.
After isolating the pellets, wash the cells again to the twice washed cell pellets resus. Suspend them in 10 milliliters of ice cold RPMI with 5%FCS and repeat the centrifugation after the spin pour off the supernatants. Then pool all the populations together into a new 10 milliliter tube with six milliliters of ice cold RPMI containing 5%FCS sediment, the pooled cells by centrifugation and aspirate off the SUP natant with a transfer pipette at this point of the whole procedure.
Six intra assay replicates can be generated by labeling the peptide pulse cells with six different concentrations of CPD. First, add 11.4 milliliters of room temperature RPMI containing 5%FCS to the pooled cell pellet and resuspend thoroughly by pipetting. Second, add 1.9 milliliters of cell suspension to six new 10 milliliter tubes labeled A to F, taking care not to wet the top half of the tube.
To start labeling the cells with CPD uncapped tube B and lay the tube horizontally along the dry wall First, add 92 microliters of PBS followed by a volume of stock CPD according to table four. Then vortex the mixture. Repeat this step with tube C two F ensuring each tube gets a different CPD stock as in table four.
After all the tubes are mixed, allow them to incubate for five minutes at room temperature. Then wash the cells twice first resuspend them in 10 milliliters of room temperature media. Second, spin them down and third aspirate off the supernatants with a transfer pipette.
Then repeat the process. Now pool all cells from tubes A to F together into a single tube in eight milliliters of ice. Cold media.
Collect the cells by spinning them down and proceed with injection of the cells into animals and subsequent flow cytometry analysis. Details are given in the text protocol. A BC mouse was immunized with recombinant vaccinia virus expressing HIV V one epitopes and responses to various epitopes were assessed using a 252 parameter FTA assay.
Two epitope variants gag mute and FTL mute were not expressed in the vector and naive mice were also injected with the FTA. The TA was discriminated from host mouse cells by P KH 26 labeling and FTAB cells were discriminated by B two 20 antibody staining via flow cytometry. Each of the six intra animal replicates were gated based on CPD fluorescence FTA targets expressing the various concentrations of epitopes were gated based on CFSE and CTV fluorescence.
The fraction of specific killing of each replicate was assessed by comparing FTA cluster cell death in primed animals relative to corresponding FTA clusters in naive animals. Helper T activity was assessed by comparing FTA B-cell upregulation of CD 69 in primed animals relative to corresponding FT a B-cell clusters in naive animals. From this analysis, V-V-H-I-V infection generated the strongest CTL responses against the immunodominant VV epitope F two L with 100%of FDA target cells pulsed with 0.001 micromolar or more of the epitope being removed from the spleen.
In addition to CTL responses, the FTA assay also measured helper T responses by assessing activation of FTAB cells, expressing the H-I-V-M-H-C class two binding epitope HIV gag th This showed that antigen specific B-cell activation occurred in primed animals suggesting a generation of HIV gag, T specific helper T-cell effectors. After watching this video, you should have a good understanding of how to generate a fluorescent target array of over 250 target cells.
