An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The overall goal of this procedure is to expand in variant natural killer T or INKT cells harvested from the mouse spleen. This is accomplished by first isolating splenic mononuclear cells, followed by enrichment of the CD five positive lymphocyte population by magnetic cell separation. The splenic INKT cell fraction is then further isolated by fluorescence activated cell sorting.

In the final step, the sorted INKT cells are seated for expansion in vitro. Ultimately, the cytokine profile of the expanded INKT cells can be assessed by Eliza. Generally, individuals new to this method will struggle as the purification and expansion of INKT cells requires the combination of different techniques performing.

This procedure will be shrogen a grad student from my laboratory. After harvesting the spleen, transfer the tissue into a 70 micron nylon filter, then use a two milliliter syringe plunger to gently mash the spleen through the strainer into a 50 milliliter conical tube and wash the filter with five milliliters of PBS. Then add three milliliters of the coal pack to a 15 milliliter tube and overlay the density gradient with the five milliliters of cellular suspension.

Separate the cells by centrifugation and transfer the interface to a new 15 milliliter tube. Then wash the cells in 10 milliliters of cold PBS and resuspend the pellet in two milliliters of fax buffer to enrich for the CD five positive splenic lymphocytes. Adjust the cell dilution to one times 10 to the seventh cells per 90 microliters with more fax buffer and add 10 microliters of anti CD five microbeads to the cells.

After 15 minutes at four degrees Celsius, wash the cells with four milliliters of fax buffer and resuspend the pellet in 500 microliters of fresh fax buffer. Isolate the CD five positive cells by magnetic cell separation according to the manufacturer's recommendation. Then dilute the enriched CD five positive lymphocytes at one times 10 to the six cells per 20 microliters inax buffer containing anti CD 16, CD 32, and PE conjugated alpha gals air CD 1D tetramer at room temperature in the dark after 30 minutes, incubate the cells with the antibodies of interest at four degree Celsius in the dark for another 30 minute.

Then wash the cells in fresh fax buffer and stain them with 10 microliters of a 20 micromolar working solution of DPI per one times 10 to the six cells for five minutes. At room temperature now acquire approximately one times 10 to the fourth enriched CD five positive lymphocyte events on the flow cytometer electronically gating on the forward scatter with low cells to exclude the cell doublets and aggregates electronically. Gate out the DAPI positive CD eight positive CD 19 positive cells within the forward scatter with low population and use side scatter and forward scatter area plots.

To electronically select the lymphocytes plot the alpha gal SER CD 1D tetramers by CD three epsilon and electronically ga the alpha gal ser CD 1D tetramer positive CD three epsilon positive INKT cells as indicated. Then acquiring no more than two times 10 to the fifth CD five positive enriched lymphocytes determine the percentage of alpha gal SER CD 1D tetramer positive CD three epsilon positive INKT cells present in the enriched cellular fraction. Using this gate low sort, the alpha alser CD 1D tetramer positive CD three epsilon positive INKT cells into a fax tube containing one milliliter of fetal calf serum at the end of the sort.

Rinse the isolated NKT cells from the side of the fax tube with 10 milliliters of RPMI 1640 medium. Then spin down the cells, resuspend the pellet in one milliliter of RPMI 1640 and use a 40 microliter aliquot of the cells to assess the purity of the sorted cells. Using the same gating strategy.

As for setting up the sort parameters to expand the INKT cells, spin down the cells and resuspend the isolated cells at five times 10 to the fifth cells per milliliter in complete RPMI 1640 medium supplemented with IL two IL 12, and anti CD 28. Then seed the cells at one times 10 to the fifth INKT cells per well in a 96 well flat bottomed anti CD three epsilon coated plate and incubate them at 37 degree Celsius in a cell culture incubator. After two days, transfer the cells to a fresh uncoated flat bottom 96 well plate for culture under resting conditions in complete RPMI 1640.

Medium for another two day incubation. On the fourth day after sorting, gently pipette the supernatant in each well to dislodge the cells and pool them in a 15 milliliter conical tube. After centrifugation, re suspend the palate at five times 10 to the fifth cells per milliliter.

In complete RPMI 16 medium supplemented with IL seven and seed one times 10 to the fifth cells per well. In a new flat bottomed 96 well plate return the cells to the incubator after four days, pull them in a fresh 15 milliliter conical tube for centrifugation. Re suspend the pellet in complete RPMI 1640 medium containing soluble anti CD 28 and seed the cells onto a 96 well flat bottom plate coated with anti CD three epsilon at one times 10 of the fifth cells per well and return the cells to the incubator for another three days, four days, 11 through 18.

Repeat the IL seven anti CD 28 and anti CD three epsilon treatment as just demonstrated on day 19. Transfer the cells to a new 15 milliliter conical tube. Spin them down and resuspend the pellet at five times 10 to the fifth cells per milliliter in complete RPMI 1640.Medium.

Then seed the cells at one times 10 to the fifth cells per well in a flat bottom 96 well plate and allow the cells to rest for two days in the cell culture incubator. Stimulating sorted INKT cells with plate bound anti CD three epsilon in the presence of IL two IL 12 and soluble anti CD 28 results in an approximately threefold expansion of INKT numbers following two days of culture. Subsequent expansion of the INKT cells in the presence of IL seven results in a three to fourfold increase in the number of INKT cells present at day four in culture.

Notably repeating these culture conditions can yield an average of seven times 10 to the seventh INKT cells after two rounds of expansion without any visible loss of cells between the changes in culture media on different days, resulting in an at least 70 fold expansion in 18 days. Further expanded INKT cells activated with plate bound recombinant murine. CD 1D loaded with alpha alser produce IL two IL four IFN gamma, TNF alpha and IL six at 48 hours post stimulation, demonstrating that the INKT cells retain their ability to produce TH one and TH two cytokines post expansion.

While attempting this procedure, it is important to remember that the proper staining of INKT cells with alpha ceramide loaded cdre is essential to identify the INKT cells while flow cytometry.Sure.