TITLE: Combined Single-Cell Measurement of Cytokine mRNA and Protein in Immune Cells
AUTHORS: Julian J. Freen-van Heeren, Benot P. Nicolet, Monika C. Wolkers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">A key feature of immune cells, such as T cells, is their rapid responsiveness to activation. The response rate of T cells depends on the type of signals they receive, and the signal strength. Studying the underlying mechanisms that define responsiveness, however, is confounded by the fact that immune cells do not uniformly respond to activation. Tools that measure gene products on a single cell level therefore provide additional insights in T cell biology. Here we describe Flow-FISH, a high-throughput assay that allows for the simultaneous measurement of cytokine mRNA and protein levels of the gene(s) of interest by flow cytometry. We present several possible applications of Flow-FISH in human and murine T cells that - with minor adjustments - should also be applicable for other gene products and cell types.  </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-style:italic;">This is a post-peer-review, pre-copyedit version of an article published in Methods in Molecular Biology, and is currently in press.</span></div></div></div>

[STEPS]
?. [1. Stimulation and extracellular staining of human and murine CD8+ T cells]
Activate 3 x 105 [human] or 1 x 106 cells [murine] in 200 L medium containing the stimulus of choice and 2 µM monensin in a sterile 96-well plate according to standard operating procedure or as described in [29, 31]. Include a sample without the stimulus as control. Cover the plate with a lid and culture cells for the desired amount of time in a humidified incubator at 37 ºC + 5% CO2 (see Notes 1 - 4).
?. [1. Stimulation and extracellular staining of human and murine CD8+ T cells]
Remove plate from incubator. Depending on the preference of the experimenter, samples can be transferred into tubes now or at step 3.2.3 [human]/3.3.6 [murine]. Otherwise, perform surface antibody staining in the stimulation plate. In case plate-bound antibodies were used for activation, transfer cells to tubes or to a 96-well V-bottom plate. Spin for 3 min at 650 x g, maximum acceleration and brake, at 4 ºC. Of note, all subsequent centrifugation steps are all performed with maximum acceleration and brake. Remove supernatant.
?. [1. Stimulation and extracellular staining of human and murine CD8+ T cells]
Add desired surface antibodies in 30 L FACS-buffer and resuspend by pipetting. Incubate for 20 min at 4 ºC in the dark (see Notes 5 - 7).
?. [1. Stimulation and extracellular staining of human and murine CD8+ T cells]
Add 150 µL FACS-buffer and spin for 3 min at 650 x g at 4 ºC. Remove supernatant.
?. [1. Stimulation and extracellular staining of human and murine CD8+ T cells]
Add 50 µL Perm/FIX solution. Resuspend the cell pellet either by briefly vortexing or pipetting and incubate for 20-30 min at 4 ºC in the dark (see Note 10).
?. [2. Intracellular mRNA and protein staining of human CD8+ T cells]
During fixation, prepare the hybridization mix by denaturing FISH probes in 20 µL RNAse-free water. Heat to 65 ºC for 5 min in a thermocycler (see Notes 13 - 20).
?. [2. Intracellular mRNA and protein staining of human CD8+ T cells]
After cooling down to room temperature, add the intracellular antibodies to the denatured FISH probes and add 4 IU/mL RNAse inhibitor. Incubate at RT in the dark for 5 min to allow the neutralization of RNAses (see Notes 5 and 11).
?. [2. Intracellular mRNA and protein staining of human CD8+ T cells]
Prepare hybridization solution by diluting the denatured FISH probes and intracellular antibodies from step 7 in RNAse inhibitor pre-treated hybridization buffer. Adjust for the amount of samples and the final concentration of probes to be added to the sample, as determined by a probe titration assay (see Note 17). I.e., for 5 samples, add 80 L hybridization buffer to the 20 L containing 1 L of probes from step 3.2.1.
?. [2. Intracellular mRNA and protein staining of human CD8+ T cells]
After fixation, add 150 µL RNAse inhibitor pre-treated Perm/Wash solution to the cells and transfer to 1.5 mL Lo-bind Eppendorf tubes. Add 1mL RNAse inhibitor pre-treated Flow-FISH wash buffer and spin cells for 5 min at 570 x g at room temperature in a swing-out centrifuge (see Note 21).
?. [2. Intracellular mRNA and protein staining of human CD8+ T cells]
Remove supernatant while leaving a small “dead” volume of ± 50 µL, as to not disturb the cell pellet and thus limit cell loss. Add 50 µL of hybridization mix containing the FISH probes and the intracellular antibodies as prepared in step 3.2.3. Vortex 2 sec to resuspend, or scratch the bottom of the tube on a rack.
?. [2. Intracellular mRNA and protein staining of human CD8+ T cells]
Incubate overnight at 37 ºC + 5% CO2 in a humidified incubator.
?. [2. Intracellular mRNA and protein staining of human CD8+ T cells]
The following day, wash cells by adding 1 mL RNAse inhibitor pre-treated Flow-FISH wash buffer and spin cells for 10 min at 570 x g at room temperature in a swing-out centrifuge.
?. [2. Intracellular mRNA and protein staining of human CD8+ T cells]
Remove supernatant while leaving ±100 l, as to not disturb the cell pellet. Resuspend the cell pellet in the residual volume and transfer to a 5 mL polystyrene round-bottom tube and measure mRNA and protein levels on a suitable flow cytometer.
?. [3.2	Intracellular mRNA and protein staining of murine CD8+ T cells]
During fixation, pipette the required amount of intracellular antibodies in RNAse-free tubes (see Notes 5 and 12). De-activate RNAses present in the antibodies by adding 4 IU/mL RNAse inhibitor to intracellular antibodies and incubate at 4 ºC for 10 min. Denature FISH probes in 20 µL RNAse-free water by heating to 65 ºC for 5 min in a thermocycler (see Notes 13 - 20).
?. [3.2	Intracellular mRNA and protein staining of murine CD8+ T cells]
After fixation, add 150 µL RNAse inhibitor pre-treated Perm/Wash solution to the cells and spin for 3 min at 650 x g, at 4 ºC. Remove supernatant.
?. [3.2	Intracellular mRNA and protein staining of murine CD8+ T cells]
Perform intracellular staining by adding desired intracellular antibodies prepared in step 14 in 30 µL RNAse inhibitor pre-treated Perm/Wash solution and resuspend by pipetting. Incubate for 20 min at 4 ºC in the dark.
?. [3.2	Intracellular mRNA and protein staining of murine CD8+ T cells]
Wash cells with 150 µL RNAse inhibitor pre-treated Perm/Wash solution and spin cells for 3 min at 650 x g at 4 ºC. Remove supernatant.
?. [3.2	Intracellular mRNA and protein staining of murine CD8+ T cells]
Wash cells twice with 200 µL RNAse inhibitor pre-treated Flow-FISH wash buffer. Between washes, spin cells for 3 min at 650 x g, at 4 ºC. Remove supernatant.
?. [3.2	Intracellular mRNA and protein staining of murine CD8+ T cells]
Prepare hybridization solution by diluting the denatured FISH probes from step 14 in RNAse inhibitor pre-treated hybridization buffer. Adjust for the amount of samples and the final concentration of probes to be added to the sample, as determined by a probe titration assay (see Note 17). I.e., for 5 samples, add 80 L hybridization buffer to the 20 L containing 1 L of probes from step 3.3.1.
?. [3.2	Intracellular mRNA and protein staining of murine CD8+ T cells]
Resuspend cells in 50 µL RNAse inhibitor pre-treated Flow-FISH wash buffer and transfer to 1.5 mL Lo-Bind Eppendorf tube. Add 50 µL of hybridization buffer supplemented with FISH probes as prepared in step 3.3.6.
?. [3.2	Intracellular mRNA and protein staining of murine CD8+ T cells]
Incubate overnight at 37 ºC + 5% CO2 in a humidified incubator.
?. [3.2	Intracellular mRNA and protein staining of murine CD8+ T cells]
Prior to acquisition, wash cells by adding 1 mL RNAse inhibitor pre-treated Flow-FISH wash buffer and spin cells for 10 min at 570 x g at room temperature in a swing-out centrifuge (see Note 21). Remove supernatant while leaving a small “dead” volume of ±100 µL, as to not disturb the cell pellet.
?. [3.2	Intracellular mRNA and protein staining of murine CD8+ T cells]
Resuspend the cell pellet by pipetting in the residual volume and transfer to a 5 mL polystyrene round-bottom tube. Measure samples/cells on a suitable flow cytometer.
?. [1. Stimulation and extracellular staining of human and murine CD8+ T cells]
The protocol for human and murine T cells diverge here. The original protocol developed for human T cells [29] required slight alterations to preserve the protein staining of cytokines in murine T cells [31].