In vitro T cell and lymph node stromal cell co-cultures

Cell lines used:

• K14 ova LECs on P7 – express ovalbumin as self-antigen
• WT LECs on P6.

Cell numbers and seeding (day before co-culture)

• Both K14ova and WT LECs are kept in T75 flasks in culture in IDMM 10%FCS 2% PSG. Medium is removed, and flasks are washed with 10mL of PBS, 1x. 1,5 mL of trypsin are then added and cells were incubated at 37°C for 1-2 min until cells are detached from bottom, followed by 10 mL of warm medium to stop the reaction.

• Cells are transferred to a 50 mL tube, through a 70 uM filter. Flasks of the same cell line can be combined on this step.10 mL of culture medium are further used to wash the filter. Cells are spin down at 300g for 5 min at 4°C,and re-suspended  in  an  appropriate volume of culture medium for counting. Cells are counted in Burker hematocytometer, after 10x dilution in trypan blue. (This is the same for all counting steps through the protocol).

• Cells are seeded in 5x10^4 cells/well for co-culture in late afternoon (around 17.00). For seeding, the appropriate volume of the cell suspension is added to the bottom of each well in 24 well plates (usually between 10-20 ul of the original cell suspension). Culture medium is added after (1 mL per well), by carefully placing the tip of the pipetted against the well wall and letting it drop slowly on the cell suspension. Hence, cells are kept close to one another and more prone to grow. Important: culture medium is let warmed up before usage at 37C in the waterbath.

• Cells are seeded with IDMM 10% FCS, 2% PSG, 5uM of beta mercaptoethanol (B-met) (co-culture medium). B-met is filtered before being added to the culture medium (0.2 uM filter and 2mL screw syringe used)

Co-culture

RAG2-/- OT-II mice that contain naïve T cells with a T cell receptor for ovalbumin are sacrificed

Spleen digestion:

• Spleens are smashed through 70 uM filter to 50 mL collection tubes. Filters must be wet with at least 1 mL of buffer before placing the spleen. Smashing is performed with the stick of 1mL syringe, gently. A filter is used for each spleen and washed with at least 10 mL of buffer during/after digestion. More buffer can be used depending on visual evaluation of the filter. 3-5 spleens are combined in each tube.
• Buffer: PBS 2%FCS
• Collection tubes are spin down at 300g for 7 min at 4 degrees. Supernatant discarded.
• Pellets were resuspended in ACK lysis buffer. ACK buffer must be at room temperature before being added to the cells. 5 mL of buffer are used per 4-5 spleens, and the tubes are kept at room temperature for 5 min after resuspension.
• Lysis is stopped with addition of 10 mL of washing buffer. Collection tubes are spin down at 300g for 7 min at 4 degrees. Supernatant is discarded.
• Cells are resuspended in selection buffer.
• Selection buffer used: PBS 2% FCS 5mm EDTA
• Count cells
   

Isolation of CD4+ cells from splenocyte mix:

• Collection tubes are spin down at 300g for 7 min at 4 degrees. Supernatant was discarded.
• Cells are ressupensed in 1x10^7 cells/100 ul as recommended by the selection kit used. (Magni Sort Enrichment kit from Thermofisher) in 5mL polycystrene tube. Note: We don’t use more than 1500 ul per tube. We split cells through several tubes if needed.
• Thermofisher protocol is then followed:
  • Add 20 µL of MagniSortTM Enrichment Antibody Cocktail per 100 µL of cells. Mix well by pulse vortexing 5 times. Incubate at room temperature for 10 minutes:
  • Wash cells by bringing the volume up to 4 mL with separation buffer and then centrifuge at 300 x g for 5 minutes. Discard the supernatant and thoroughly resuspend the cells to their original volume with separation buffer.
  • For cells from spleen only, add 20 µL of MagniSort TM Negative Selection Beads per 100 µL of cells. Incubate at room temperature for 5 minutes.
  • Bring the volume up to 2.5 mL with desired cell separation buffer. Mix by pipetting up and down 3 times with a P1000 pipette set to 1 mL. Avoid vortexing.

  • Insert the tube into the magnet until the bottom of the tube is touching the bench top through the hole in the bottom of the magnet. Incubate at room temperature for 5 minutes.

  • Pick up the magnet, and in a continuous motion pour the supernatant into a 15 mL conical tube. Hold the inverted tube for 1 second and then return it to the upright position.
• Cells are resuspended in selection buffer in total of 10 mL.
• Count cells.

Distribution of T cells:

• Cells are distributed at 0.5 x 10^6 cells/well. Each culture condition is a combination of 4-6 wells.
• Around 0.1 to 0.2 x 10^6 cells should be transferred for 96-well plate to perform a small input staining (the information on the T cell profile before co-culture).
• OT-II peptide is added in desired concentration when T cells are already in culture, around 2hr after T cell transfer.

Input staining:

• Ressuspend cells in 100 ul (1:2000 dilution) of fixable APC-ef780 Life Death staining for 15 min on ice in dark.
• Wash 1x time with 100 PBS. Spin down at 300g for 4 min at 4 °C.
• Add PBS 2%FCS 5% NMS (25 ul per well) for 10 min on ice. NMS: Normal Mouse Serum.
• Add extracellular staining in PBS 2%FCS 5% NMS (25 ul per well, total stained in 50ul). Incubate for 30 min on ice in dark
• Wash 2x with 150 ul PBS 2% FCS.
• Add 4x diluted ebio fix buffer (as per fabricant instructions), 100 ul per well. Incubate for 45 min on ice in dark.
• Wash with 100ul 1x with ebio perm (10x diluted in water). Spin down at 300g for 4 min at 4 °C.
• Add intracellular staining in 50 ul per well. Abs must be diluted in 1x ebio perm buffer. Incubate for 1hr on ice in dark.
• Wash 1x with 150 ul 1x ebio perm.  Spin down at 300g for 4 min at 4 °C
• Wash 1x with 200 ul  PBS 2% FCS. Spin down at 300g for 4 min at 4 °C
• Wash 1x with 200 ul PBS. Spin down at 300g for 4 min at 4 °C
• Transfer to FACs tubes (50 ul per condition in PBS).

Example of extracellular Staining:

Example of intracellular Staining:

Co-culture collection

After approximately, 72 hr in culture, T cells are collected for further analysis. The wells of each condition were brought together in a single replicate.

Washing process:

• Ressuspended medium carefully with 1 mL pipette. After at least five ressuspensions, remove the the medium to a 50 mL tube through a 70uM filter. Make sure the filter is wet before adding cells. Repeat the ressuspension for every well of the same condition collect them through the same filter. 4-6 wells are combined together in this step.

• Add 1 mL of cold PBS 2% FCS to the wells. Ressuspend each well at least five times and collect through the same filter. Repeat the washing with the buffer a total of three times.

• When finished, wash the filter with 10 to 15 mL of cold PBS 2% FCS carefully.
• Repeat it to every condition. Keep the cells on ice while collecting other conditions.
• After collection, spin cells down for 7 min at 300 g at 4°C. Discard supernatant.
• Ressuspend cells carefully in an appropriate volume for counting.
• Add 10 mL of cold PBS 2% FCS to all tubes.
• Spin down 7 min at 300 g at 4°C. Discard supernatant as carefully as possible. Use leftover volume (add more buffer only if needed) to resuspend the pellet carefully with a yellow tip pipette. Transfer the cells to a 96-well plate.
• Spin down 4 min at 300g and start staining. The procedure is the same as decribed for input staining above, including staining volumes given cell numbers are fairly low.

Example of extracellular staining:

Example of intracellular staining

Consider taking FMOs for lowly expressed genes. In our example, we have included FMOs for CD25, FoxP3, CXCR5, IL-2 and BCL-6.

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