TITLE: Influenza A Virus-Infected Lung Epithelial Cell Co-Culture with Human Peripheral Blood Mononuclear Cells
AUTHORS: Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Timothy S. C. Hinks

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sensing of influenza A virus (IAV) infection by pattern recognition receptors can occur by either direct infection of lung epithelial cells or uptake of virus-infected cells by innate cells such as dendritic cells/monocytes. This triggers a series of downstream events including activation of the inflammasome, the production of cytokines, chemokines, and the upregulation of stress-induced ligands that can lead to the activation of innate cells. These cells include innate lymphocytes such as MAIT, NKT, NK, and γδ T cells. Here we describe a method used to allow activation of human innate lymphocytes in co-culture with an IAV-infected human lung epithelial cell line (A549) to measure ex vivo effector functions (TNF and IFNγ) in a mixed culture environment. We describe (1) infection of the human lung epithelial cell line, (2) co-culture with PBMC, and (3) measurement of activation using intracellular cytokine staining.</div></div>

[STEPS]
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
prior to infection, in two T75 flasks, seed 5 x 106 A549 cells in a total volume of .
[media]
One flask for IAV infection and the second flask for uninfected control A549s.
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
On the day of infection:
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Leave one flask of A549 cells in the incubator (uninfected control).
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Wash the other flask with  once, cap and gently rotate flask from side to side. Aspirate PBS with glass tissue culture pipette.
[PBS]
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Thaw virus (PR8)  and add  to  in a 50 mL falcon tube (depending on viral titer of stock) to achieve a multiplicity of infection (MOI) of ~10–30. Gently pipette this into the T75 containing A549 cells.
on ice
[virus]
[room temperature PBS]
Example calculation of MOI 10: An MOI of 10 using 1x106 PBMC per well requires 1x107 virus particles/well. The volume required/well of a 1x109 plaque forming units (pfu)/mL virus titer is:1x107 pfu/1x109 pfu/mL = 0.01 mL/well or 10 μL/well.
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Incubate flask horizontally for  in the  incubator (5% CO2).
37 °C
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Remove both T75 flasks from incubator and add  to the flask containing virus. Cap and gently rotate from side to side. Aspirate media from both flasks.
[cRPMI]
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
To detach A549 cells, wash flasks once with , aspirate, and add  to each flask. Gently tilt the flask to ensure that the solution coats the entire flask.
[PBS]
[Trypsin versene]
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Incubate for  in the  incubator (5% CO2).
37 °C
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Add  to T75 flasks and transfer the contents into two 50 mL falcon tubes.
[cRPMI]
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Aspirate supernatant.
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Resuspend cells in  and perform cell counts using trypan blue estimation.
[cRPMI]
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Adjust the volume of A549 cells so that the final concentration is 2x106 cells/mL.
?. [IAV Infection of Human Lung Epithelial Cell Line, A549]
Centrifuge .
Centrifuge: 500 34, 5 min, 25 10
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Thaw PBMCs in .
[water bath]
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Gently pipette dropwise into  per cryovial.
[pre-warmed cRPMI]
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Centrifuge .
Centrifuge: 500 34, 5 min
MAIT cell responses after in vitro influenza co-culture are highly variable between donors. Freshly processed PBMCs may aid in the detection of IFNγ cytokine responses after influenza co-culture.
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Aspirate media and count cells.
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Resuspend PBMCs at 10x106 cells/mL in cRPMI.
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
For each sample, aliquot  into three wells of a 96-well U-bottom plate.
[cells (1x10^6 PBMC)]
These wells will correspond to Media Control, uninfected A549 + PBMC, and IAV-infected A549 + PBMC, respectively.
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Check IAV nucleoprotein levels:
To determine if influenza virus infection of lung epithelial cells is successful after 10 h of culture, intracellular cytokine staining for influenza A virus nucleoprotein is determined by flow cytometry. (Steps 25-27 followed by steps 31-38, shown here in subsequent substeps for convenience)
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Add  of uninfected A549s or IAV-infected A549s (2x105 cells) into wells containing PBMC. Leave one well with PBMC only, add  to this well. Place plate in the  incubator (5% CO2).
100 µl
[cRPMI]
37 °C
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
After –, add brefeldin A (BFA-GOLGI PLUG), 1:2000 to all wells.
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Incubate for a further  in the  incubator ().
37 °C
[total co-culture]
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Remove plate and continue with intracellular cytokine (ICS) staining or place in the  covered in foil to stain the next day.
4 °C
?. [Intracellular Cytokine Staining]
Spin down plate by centrifuging at . Discard supernatant in waste container containing 10% Lysol or 1% Virkon in class II biosafety cabinet.
Centrifuge: 400 34, 5 min, 4 10
?. [Intracellular Cytokine Staining]
Stain cells with live/dead discrimination marker Aqua (1:800) final volume of 50 μL/well. Use PBS as a diluent. Incubate at  in the dark for .
4 Room temperature
Fixable viability dyes react with exposed amine groups within permeable cells. Therefore, to prevent wasteful reaction with proteins in cytometry buffers, it is recommended to resuspend cells in protein-free media for the viability staining step.
?. [Intracellular Cytokine Staining]
Centrifuge plate at . Discard supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Intracellular Cytokine Staining]
Add  (Table 1 below) to each well. Table 1 (Antibodies):Example flow cytometric activation panel and IAV nucleoprotein expression   ABCDE1MarkerFluorophoreLaserCloneDilution2Surface stain activation3Live DeadAquaViolet1/800 (stain in PBS prior to surface stain)4MRI-5-OP-RU TetSA-BV421Violet*Titrate 1/200-1/4005CD19APC-H7RedH1B191/1006CD14APC-H7RedMΦP91/1007CD8αPerCP-Cy5.5BlueSK11/508TCRγδFITCBlue2F111/509CD4BV650VioletOKT41/20010CD161BV605VioletHP-3G101/5011CD3PE-CF594Yellow/GreenUCHT11/20012TCR Vα7.2PEYellow/Green3C101/20013CD56PE-Cy7Yellow/GreenNCAM16.21/10014Intracellular stain activation15TNFAPCRedMAb111/5016IFNγAF700RedB271/15017Intracellular stain IAV-NP infection18IAV nucleoproteinFITCBlue13311/100
[surface phenotype stain]
ABCDE1MarkerFluorophoreLaserCloneDilution2Surface stain activation3Live DeadAquaViolet1/800 (stain in PBS prior to surface stain)4MRI-5-OP-RU TetSA-BV421Violet*Titrate 1/200-1/4005CD19APC-H7RedH1B191/1006CD14APC-H7RedMΦP91/1007CD8αPerCP-Cy5.5BlueSK11/508TCRγδFITCBlue2F111/509CD4BV650VioletOKT41/20010CD161BV605VioletHP-3G101/5011CD3PE-CF594Yellow/GreenUCHT11/20012TCR Vα7.2PEYellow/Green3C101/20013CD56PE-Cy7Yellow/GreenNCAM16.21/10014Intracellular stain activation15TNFAPCRedMAb111/5016IFNγAF700RedB271/15017Intracellular stain IAV-NP infection18IAV nucleoproteinFITCBlue13311/100
?. [Intracellular Cytokine Staining]
Wash cells once with . Centrifuge for . Flick off supernatant in discard container in biohazard cabinet.
[FACs buffer]
Centrifuge: 1500 33, 5 min, 4 10
?. [Intracellular Cytokine Staining]
Wash cells with . Centrifuge for .
[ diluted (1:10 in dH2O) perm/wash buffer]
Centrifuge: 450 34, 5 min, 4 10
?. [Intracellular Cytokine Staining]
Resuspend cells in , see (Table 1) below. Table 1 (Antibodies):Example flow cytometric activation panel and IAV nucleoprotein expression    ABCDE1MarkerFluorophoreLaserCloneDilution2Surface stain activation3Live DeadAquaViolet1/800 (stain in PBS prior to surface stain)4MRI-5-OP-RU TetSA-BV421Violet*Titrate 1/200-1/4005CD19APC-H7RedH1B191/1006CD14APC-H7RedMΦP91/1007CD8αPerCP-Cy5.5BlueSK11/508TCRγδFITCBlue2F111/509CD4BV650VioletOKT41/20010CD161BV605VioletHP-3G101/5011CD3PE-CF594Yellow/GreenUCHT11/20012TCR Vα7.2PEYellow/Green3C101/20013CD56PE-Cy7Yellow/GreenNCAM16.21/10014Intracellular stain activation15TNFAPCRedMAb111/5016IFNγAF700RedB271/15017Intracellular stain IAV-NP infection18IAV nucleoproteinFITCBlue13311/100
[intracellular cytokine stain]
ABCDE1MarkerFluorophoreLaserCloneDilution2Surface stain activation3Live DeadAquaViolet1/800 (stain in PBS prior to surface stain)4MRI-5-OP-RU TetSA-BV421Violet*Titrate 1/200-1/4005CD19APC-H7RedH1B191/1006CD14APC-H7RedMΦP91/1007CD8αPerCP-Cy5.5BlueSK11/508TCRγδFITCBlue2F111/509CD4BV650VioletOKT41/20010CD161BV605VioletHP-3G101/5011CD3PE-CF594Yellow/GreenUCHT11/20012TCR Vα7.2PEYellow/Green3C101/20013CD56PE-Cy7Yellow/GreenNCAM16.21/10014Intracellular stain activation15TNFAPCRedMAb111/5016IFNγAF700RedB271/15017Intracellular stain IAV-NP infection18IAV nucleoproteinFITCBlue13311/100
?. [Intracellular Cytokine Staining]
Wash cells with . Centrifuge for .
[perm/wash buffer]
Centrifuge: 450 34, 5 min, 4 10
?. [Intracellular Cytokine Staining]
Resuspend cells in  and transfer to bullet tubes.
[1% PFA]
Keep samples in the dark and at cold until acquisition on the flow cytometer.
?. [Intracellular Cytokine Staining]
Incubate for , in the dark.
on ice
?. [Intracellular Cytokine Staining]
Resuspend the cells in  and incubate  in the dark for .
[cold cytofix/perm solution]
on ice
?. [Intracellular Cytokine Staining]
Incubate   in the dark for .
on ice
?. [Intracellular Cytokine Staining]
Repeat with a second wash with . Centrifuge for .
[FACs buffer]
Centrifuge: 450 34, 5 min, 4 10
?. [Intracellular Cytokine Staining]
For suggested flow cytometric gating strategy see Fig. 1. below:
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Spin down plate by centrifuging at . Discard supernatant in waste container containing 10% Lysol or 1% Virkon in class II biosafety cabinet.
Centrifuge: 400 34, 5 min, 4 10
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Stain cells with live/dead discrimination marker Aqua (1:800) final volume of 50 μL/well. Use PBS as a diluent. Incubate at  in the dark for .
4 Room temperature
Fixable viability dyes react with exposed amine groups within permeable cells. Therefore, to prevent wasteful reaction with proteins in cytometry buffers, it is recommended to resuspend cells in protein-free media for the viability staining step.
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Centrifuge plate at . Discard supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Resuspend the cells in  and incubate  in the dark for .
[cold cytofix/perm solution]
on ice
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Wash cells with . Centrifuge for .
[ diluted (1:10 in dH2O) perm/wash buffer]
Centrifuge: 450 34, 5 min, 4 10
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Resuspend cells in , see (Table 1) below. Table 1 (Antibodies):Example flow cytometric activation panel and IAV nucleoprotein expression    ABCDE1MarkerFluorophoreLaserCloneDilution2Surface stain activation3Live DeadAquaViolet1/800 (stain in PBS prior to surface stain)4MRI-5-OP-RU TetSA-BV421Violet*Titrate 1/200-1/4005CD19APC-H7RedH1B191/1006CD14APC-H7RedMΦP91/1007CD8αPerCP-Cy5.5BlueSK11/508TCRγδFITCBlue2F111/509CD4BV650VioletOKT41/20010CD161BV605VioletHP-3G101/5011CD3PE-CF594Yellow/GreenUCHT11/20012TCR Vα7.2PEYellow/Green3C101/20013CD56PE-Cy7Yellow/GreenNCAM16.21/10014Intracellular stain activation15TNFAPCRedMAb111/5016IFNγAF700RedB271/10017Intracellular stain IAV-NP infection18IAV nucleoproteinFITCBlue13311/100
[intracellular cytokine stain]
ABCDE1MarkerFluorophoreLaserCloneDilution2Surface stain activation3Live DeadAquaViolet1/800 (stain in PBS prior to surface stain)4MRI-5-OP-RU TetSA-BV421Violet*Titrate 1/200-1/4005CD19APC-H7RedH1B191/1006CD14APC-H7RedMΦP91/1007CD8αPerCP-Cy5.5BlueSK11/508TCRγδFITCBlue2F111/509CD4BV650VioletOKT41/20010CD161BV605VioletHP-3G101/5011CD3PE-CF594Yellow/GreenUCHT11/20012TCR Vα7.2PEYellow/Green3C101/20013CD56PE-Cy7Yellow/GreenNCAM16.21/10014Intracellular stain activation15TNFAPCRedMAb111/5016IFNγAF700RedB271/10017Intracellular stain IAV-NP infection18IAV nucleoproteinFITCBlue13311/100
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Incubate  in the dark for .
on ice
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Wash cells with . Centrifuge for .
[perm/wash buffer]
Centrifuge: 450 34, 5 min, 4 10
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Repeat with a second wash with . Centrifuge for .
[FACs buffer]
Centrifuge: 450 34, 5 min, 4 10
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Resuspend cells in  and transfer to bullet tubes.
[1% PFA]
Keep samples in the dark and at cold until acquisition on the flow cytometer.
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
For suggested flow cytometric gating strategy see Fig. 1. below:
?. [Co-Culture (Start During the 1 h Incubation with Virus)]
Add  of infected and uninfected A549 cells to separate wells in the 96-well plate.
100 µl