Analyzing efficiency of a lentiviral shRNA knockdown system in human enteroids using western blot and flow cytometry

Summary Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes.


Highlights Protocol for lentivirus-based gene editing of gastrointestinal enteroids
Steps for performing a puromycin selection trial Instructions for assessing transduction efficiency Note: Enteroids characterized by spheroid morphology (Figure 1A) are optimal for both puromycin selection cytotoxicity trials and lentiviral transfection.Highly budded (differentiated) enteroids (Figure 1B) proliferate and propagate at reduced rates following transfection.In our hands, optimal morphology occurs approximately 2 days post-passaging, resulting in enteroids that are no larger than 100-150 mm in diameter.
Note: Allow Matrigel to thaw on ice at 4 C for at least 12 h.Cool phosphate-buffered saline (PBS) and DMEM/F-12 (Dulbecco's Modified Eagle Medium F12) at 4 C for at least 12 h.Note: All steps involving viable cells should take place within a sterile biological safety cabinet (here, we use a Type A2, but any appropriately rated and sterile cabinet will work).

Microplate Matrigel coating (Troubleshooting).
a. Place a CoolSink XT 96F on ice a minimum of 30 min before plating.b.Once ice-cold, place a sterile 96-well microplate on the CoolSink XT 96F.c.Using a 200 mL pipette and pipette tips, add 100 mL ice-cold PBS to each experimental well.Allow PBS to sit in wells for 1 min.d.After 1 min, remove PBS from each well and replace with 30 mL ice-cold Matrigel.e. Place plate in 37 C incubator for a minimum of 20 min to solidify Matrigel.Retain plate in incubator until ready to plate cells.
Alternative: We use a Heracell VIOS CO 2 incubator, but any sterile incubator containing a humidity reservoir, high-efficiency particulate air (HEPA) filtration, heat chamber, and CO 2 capabilities will work.

Passage enteroids through dissociation into single cells (Troubleshooting).
Note: TrypLE Express is used in lieu of trypsin.TrypLE is formulated with high purity, eradicating extraneous enzymes often present in trypsin, eliminating the need for fetal bovine serum (FBS) inactivation of the enzyme, and reducing cell damage during passaging.
Note: TrypLE Express should be warmed to 37 C in a water or bead bath prior to use.

Note:
The number of wells of a 24-well plate required to seed a 96-well plate will depend upon enteroid line growth rates and characteristics of source tissue.The following steps are calibrated for use of 6 wells of a 24-well plate, equating to 1 3 10 5-6 cells/well with the IL1004 enteroid line.
b. Using a 1 mL pipette and pipette tips, add 500 mL of prewarmed TrypLE Express to each well.c.Mechanically disrupt Matrigel domes by pipetting up and down 5 times.d.Place plate in 37 C incubator for 10 min.Further mechanical disruption may be required after 10 min to break up remaining cell clusters.
CRITICAL: To prevent cell death, do not leave enteroids in TrypLE Express longer than 10 min.
e. Following the 10 min incubation, add 500 mL ice-cold DMEM/F-12 to each well to dilute TrypLE Express.f.Once enteroids are dissociated to single cells, place contents of wells into a 15 mL centrifuge tube.g.Centrifuge at 400 3 g at 4 C for 5 min to pellet cells.

Note:
We use an Eppendorf multipurpose swing bucket rotor centrifuge with adapters fit to multiple tube sizes, but any centrifuge with appropriate capabilities will suffice.
Note: Cells (white) will collect at the bottom of the 15 mL centrifuge tube.Remaining Matrigel will form an opaque layer immediately above the cell pellet.At 4 C, Matrigel liquifies and should be removable through aspiration or careful pipetting.h.Remove supernatant containing Matrigel, resuspend pellet in 4 mL DMEM/F-12, and centrifuge again at 400 3 g and 4 C for 5 min.i. Remove all but 500 mL of supernatant.
Note: If Matrigel is still visible around the cell pellet, repeat ice-cold DMEM/F-12 washes until Matrigel completely dissolves and is removed.

Count cells for consistent and even plating (Troubleshooting).
Alternative: We utilize the automated Countess 3 cell counter, but any automated cell counter system or manual hemocytometer will suffice.a. Resuspend pellet in remaining 500 mL of DMEM/F-12.b.Using a 20 mL pipette and pipette tips, add 15 mL of cell suspension into a 1.50 mL microcentrifuge tube.c.Add 15 mL 0.40% trypan blue stain and mix thoroughly via pipette.d.Pipette 10 mL of stained cell suspension into each of the two chambers of a Countess cell counting chamber slide.e. Insert one end of the chamber slide into the Countess 3, and record number and viability of cells.f.Repeat measurements with second slide chamber.
Note: Our laboratory has standardized cell concentrations for a 96-well plate to 6 3 104 cells/ mL, but optimal plating concentrations may vary.4. Plate enteroid single-cell suspension in Matrigel sandwich.
a. On day 0, calculate the total volume of WRNE media required to resuspend cells such that 100 mL of final cell suspension will seed each well of the 96-well plate with an empirically determined cell density (see above Note).

Note: Cell pelleting (as in Preparation
Step 2g) may be required if tube media already exceeds target volume.
b. Remove 96-well microplate from incubator.c.Pipette 100 mL of single-cell suspension onto Matrigel layer within each experimental well.d.Return plate to incubator for 24 h.e.The following day (day 1), gently aspirate media from each well, leaving only cells attached to the bottom layer of Matrigel.f.To finalize the Matrigel sandwich, overlay 30 mL of Matrigel on attached cells.g.Place microplate in a 37 C incubator for 20 min.h.Following 20 min incubation, add 100 mL of WRNE media (20 C-22 C) to each experimental well.
Note: Media should be dispensed into wells against the sidewalls, slowly and gently, so as not to disturb the Matrigel sandwich.
Note: Allocate sufficient wells to each treatment so as to be powered to make definitive conclusions at trial end.
Note: Cells should be visualized daily via bright-field microscopy for general morphology to aid in determining puromycin dosing duration.
Alternative: Our laboratory uses a Revolution Hybrid automated microscope, but any microscope with 10X or 20X bright-field capabilities and microplate adaptors will be sufficient.
b.In 2 days (day 4), replace culture media with fresh WRNE + puromycin.c.Repeat Preparation Step 5b, with the last WRNE + puromycin change occurring at day 6.d.In 3 days (day 9), determine cell viability through cell counting or commercially available viability assays.e.The minimum concentration of puromycin resulting in complete cell death after 5-9 days of selection represents the optimal concentration for this cell type during transfection.
Note: This concentration may also be effective over a shortened dosing period, but dosing duration should be empirically determined by each laboratory.This optimal puromycin concentration will be used to select cells for downstream applications (e.g., western blotting, Steps 6-16).

Timing: 30 min
This step describes calculations required for determining lentiviral titer, multiplicity of infection (MOI), and transducing units.Because the relationship between reported lentiviral titer (determined by particle vendor through p24 enzyme-linked immunosorbent assay [ELISA]) and functional titer is dependent upon the viral vector and cell type to be transfected, functional titer should be optimized by each laboratory.Particularly difficult cell lines may require an MOI up to 50-100.Both the flow cytometry and western blot protocols described below are suitable in determining optimal MOI/ functional titer per vector and cell line.Optimization in our laboratory for enteroid transfection has resulted in an MOI of 20.
Alternatives: Techniques such as colony forming unit (CFU) assays, fluorescence-activated cell sorting (FACS) titration, or quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) are additional commonly used techniques for optimizing functional titers.
Note: Transduction efficiency can be optimized using the MISSION pLKO.1-puro-CMV-tGFPpositive control transduction particles before progressing to use of targeted short hairpin ribonucleic acid (shRNA) constructs.This is a critical step in differentiating effects of a particular gene knockdown from those of transduction efficiency, should protocol troubleshooting be required.
Note: In addition to viral particles targeted for knockdown of the gene of interest, appropriate control particles should also be run with each experiment.Here, we utilize both negative (MISSION pLKO.1-puronon-target shRNA control transduction particles) and positive (MISSION pLKO.1-puro-CMV-tGFPpositive control transduction particles) controls.To prepare 50 mL of WRNE enteroid growth media, combine ingredients below.For preparation of large media batches, see Miyoshi and Stappenbeck 11 or VanDussen et al. 24 Our laboratory maintains L-WRN cells producing Wnt-3a, R-spondin 3, and noggin, but conditioned media generated from this cell line is also commercially available from large biomedical vendors.
To prepare $100 mL of transduction media (for 1 well of a 96-well microplate), combine the following.
Flow cytometry analysis for transduction efficiency and cell viability  To prepare 500 mL of western lysis buffer, combine the following.
Western transfer buffer.
To prepare 1 L of western transfer buffer (pH 8.3), combine the following.
To prepare 1 L of TBST with 3% BSA, combine the following.
To prepare 1 L of TBST with 5% non-fat milk, combine the following.

STEP-BY-STEP METHOD DETAILS Transfecting enteroids
Timing: 3 h This step dissociates enteroids into a single-cell suspension.Cells are then plated in a 96-well plate between two layers of Matrigel (Matrigel sandwich) for consistent access to nutrients.Transduction media contains TransDux MAX, a transduction solution similar to polybrene but with increased efficacy in primary cells.Toxicity of TransDux MAX, or similar transduction solutions, should be evaluated in cells of interest before attempting transfection, as some primary cell lines, in particular, are sensitive to these agents.
Note: Lentiviral particles are vulnerable to significant temperature shifts.Avoid freeze/thaw cycles and exposure to ambient temperatures by retaining particles on ice during use.Aliquot remaining particles into smaller working volumes and freeze at À80 C. Aliquots of lentiviral particles should always be thawed on ice.i. Seal microplate with parafilm to prevent viral contamination.j.Spinoculate plate via centrifugation at 600 3 g and 32 C for 1 h.k.Following spinoculation, verify with bright-field microscopy that cells are localized to well bottoms.l.Add 100 mL 20 C-22 C WRNE to each well, remaining careful not to disturb cells localized to bottom of wells.m.Pipette up and down to detach cells from well bottoms and transfer contents of 3 wells to a 1.50 mL microcentrifuge tube.n.Centrifuge tubes at 600 3 g at 4 C for 5 min.
Note: Our laboratory uses contents of 3 wells in a 1.50 mL microcentrifuge tube because this tube size is convenient for the refrigerated microcentrifuge we utilize (see below, Alternative).However, additional tube volumes/well numbers can be utilized, as convenient.
Alternative: Our laboratory uses a Micro 17/17R refrigerated microcentrifuge from Fisher.Centrifuge speeds depend upon the rotor radius, but any microcentrifuge with appropriate speed settings, cooling capabilities, and tube holders will suffice.o.Discard supernatant and resuspend cell pellet in 300 mL WRNE.p. Pipette 100 mL of cell suspension into each experimental well of the Matrigel-coated 96-well microplate, and place microplate in 37 C incubator for 24 h.q.The following day, aspirate media from each well, leaving only live, attached cells on bottom layer of Matrigel sandwich.r.Overlay 30 mL of Matrigel onto cells, completing the Matrigel sandwich.s.Incubate plate at 37 C and 5% CO 2 for 20 min.Note: If utilizing cells for downstream analyses (e.g., western blot), first select for transduced cells utilizing puromycin at the empirically determined concentration and dosing period resulting from Preparation Step 5e.

Flow cytometry analysis for transduction efficiency and cell viability
Timing: 6 h This step outlines the use of flow cytometry as a method for determining transduction efficiency and cell viability effects on enteroids.MISSION lentiviral particles are tagged with tGFP.Utilization of a vector incorporating tGFP allows for easy identification of transfected versus uninfected cells.In addition, the effects of lentiviral transfection on viability can be assessed through use of a fluorescent reactive dye.In dead cells, the dye penetrates compromised membranes, reacting with free amines both intracellularly and on the cell surface, producing a vibrant fluorescent staining pattern.In live cells, the dye interacts exclusively with cell surface amines, generating a reduced signal and allowing for discrimination between live and dead cells.This dye is functional on both live cells and those that have been fixed following dye administration.Note: Dye should be used within a few hours of preparation.Reconstituted dye can be aliquoted into smaller volumes and stored at À80 C for a maximum of 3 months.
c. Prepare PBS containing 1% BSA (for 10 mL, add 0.10 g BSA).Place solution on ice.CRITICAL: A minimum of 1 3 10 5 cells per condition is needed to perform flow cytometry.Pause point: Fixed, stained cells can be kept in the dark at 4 C for a maximum of 2 h.

Flow cytometry analysis (Troubleshooting steps 6 and 7).
Alternative: Our laboratory uses a BD Biosciences Accuri C6 Plus flow cytometer equipped with 488 and 640 nm lasers, but any flow cytometer with the appropriate lasers and detectors will work for this purpose.
Note: Pipette up and down several times before infusing sample into cytometer.Users may also wish to filter cell suspension with a 37 mm cell strainer to eliminate clumping immediately prior to analysis.

a. After performing quality control per manufacturer instructions, open the Accuri C6 Plus Flow
Cytometer Software, which automatically directs user to the Collect tab.b.Manually calibrate the flow cytometer by establishing compensation settings with GFP compensation beads.
Alternative: We use the BrightComp eBeads GFP Compensation Bead Kit, but stably transfected GFP-expressing cell lines can also be used for compensation.
c. Select an empty sample well and enter a sample name in the text box above the 96-well grid.
d. Set acquisition parameters to 10,000 events, slow (14 mL/min) fluidics rate, and a forward scatter (FSC) threshold between 200,000 and 500,000.A secondary threshold is set at 640 nm to eliminate background autofluorescence in the red fluorescence channel.e. GFP (fluorescein isothiocyanate, FITC) is collected in the FL1 channel (530/30) and red (phycoerythrin, PE; viability) is collected in the FL3 channel (> 670).Both are plotted logarithmically.f.Manually load the first sample over the sample injection port.g.Click run to start the sample collection and save the workspace when prompted.h.FSC vs. side scatter (SSC) dot plots are displayed by default under the Analyze tab, and gating is applied to identify single cells.i.Additional plots can be generated via altering X-and Y-axes to desired filter channels.Gating from initial plots is automatically applied to subsequent plots.j.Gating is applied to identify live and transfected (Figure 2B) cells using PE and FITC filter channels, respectively.k.Plots are exported.Raw data are also exported as .fscfiles for additional analysis.

Timing: 4 days
This step describes the use of western blotting for downstream evaluation of lentiviral gene knockdown on biological processes.
Note: Place PBS at 4 C for at least 12 h before enteroid harvest.f.Mechanically disrupt Matrigel sandwiches by pipetting up and down 5-10 times, being careful not to introduce bubbles.g.Collect contents of 3 wells in a 1.50 mL microcentrifuge tube.h.Repeat Step 6g as many times as required to collect contents from all experimental wells.i. Place microcentrifuge tubes on a tube revolver rotator set to 33 revolutions per minute (rpm) at 20 C-22 C for 15 min.
Alternative: Our laboratory uses a tube revolver rotator from Thermo Fisher Scientific, but any rotator with the appropriate speed settings and tube adaptors will work.j.After 15 min rotation, place tubes on ice for 1 h, occasionally inverting, to dissolve Matrigel.k.After 1 h of cooling, centrifuge 1.50 mL microcentrifuge tubes at 600 3 g for 5 min at 4 C. l.Aspirate supernatant and wash with 500 mL ice-cold PBS.m.Repeat Steps 6k-l until all Matrigel has been removed.n.Centrifuge tubes at 600 3 g for 5 min at 4 C. o.Remove supernatant and replace with 300 mL ice-cold western lysis buffer.p. Gently pipette up and down to resuspend cells in buffer.q.Place 1.50 mL microcentrifuge tubes on tube revolver rotator at 20 rpm for 15 min at 4 C. r.Centrifuge microcentrifuge tubes at 14,000 3 g for 15 min at 4 C to pellet cell debris.s.Transfer supernatant containing cell protein to a new 1.50 mL microcentrifuge tube and immediately flash freeze in liquid N 2 .
Pause point: Cell lysate can be stored at À80 C for up to 3 months before western blot analysis.
7. Protein quantification via BCA assay.a. Sample protein concentrations are quantified using the Pierce BCA Protein Assay Kit (microplate procedure, sample to working reagent [WR] ratio = 1:8), per manufacturer's instructions.b.Prepare diluted BSA standards (0-2000 mg/mL) in 15 mL centrifuge tubes, using western lysis buffer as diluent.c.Calculate required WR volume as: (# of standards or unknowns) x (3 replicates) x (200 mL).d.Prepare WR in a 15 mL centrifuge tube via combining BCA Reagents A and B in a 50:1 ratio.e. Pipette 25 mL of standard or sample into each well of a 96-well, clear, flat-bottom, microplate.f.Add 200 mL of WR to each well.g.Mix contents of wells thoroughly for 30 s using the plate reader shaking function.h.Cover plate with adhesive plate cover and place in a 37 C incubator for 30 min.i. Cool plate to 20 C-22 C. j.Measure absorbance on a plate reader at 562 nm.
Alternative: Our laboratory uses a SpectraMax iD3 multi-mode microplate reader, but any plate reader with the appropriate plate holders and spectral capabilities will suffice.k.Subtract the average blank 562 nm absorbance from that of all other standards and samples.l.Prepare a standard curve by plotting the average blank-corrected absorbance for each BSA standard vs. associated mg/mL concentration.
m. Use the standard curve to calculate protein concentration of each sample.8. Sample preparation for electrophoresis.
a.In 1.50 mL microcentrifuge tubes, adjust sample protein concentration to 20-25 mg/mL using 4X SDS denaturing loading buffer as a diluent.
Note: Our samples usually result in a protein concentration averaging 1 mg/mL, which we then dilute to 20-25 mg/mL with SDS denaturing loading buffer.
b. Place 1.50 mL microcentrifuge tubes in a 95 C heat block for 7-10 min.
Alternative: Our laboratory utilizes a digital dry bath/block heater from Thermo Fisher Scientific, but any similar instrument will work.
c.After 7-10 min heating, place samples on ice for 10 min to ensure complete denaturation of proteins.d.Following cooling, vortex samples briefly.
Alternative: Our laboratory uses an analog vortex mixer from Fisher Scientific, but a vortexer from any vendor with similar capabilities will work.e. Centrifuge samples at 13,500 3 g and 22 C for 2 min.9. Electrophoresis.
a. Remove the 4-20% Mini-PROTEAN TGX precast protein gel from storage pouch.b.Remove comb and green tape, and rinse wells thoroughly with 1X SDS running buffer.c.Repeat for second gel, if applicable.d.Using components of the Mini-PROTEAN Tetra Cell, 2-gel system, set the electrode assembly to the open position on a clean, flat surface.e. Place the first gel cassette (short plate facing inward, gel resting at a 30 angle away from center) onto gel supports.f.Similarly, place second gel on opposite side of clamping frame.g.Lock gels into place and create a leak-proof seal by gently pushing both gels toward each other.The edge of the short plates should sit just below the notch at the top of the green gasket.h.Gently squeeze the gel cassettes against the green gaskets.Slide green arms of the clamping frame over the gels, one at a time.i. Place the electrophoresis module into the tank.j.Fill the inner (200 mL) and outer (550 mL) buffer chambers with 1X SDS running buffer.k.Place a stir bar in outer buffer chamber to increase buffer infiltration of electrode assembly.l.Using 200 mL gel-loading tips, load 10 mL of the dual color protein ladder into the first gel lane.In each of the remaining 9 gel lanes, load 50 mL of prepared sample.m.Place lid on the Mini-PROTEAN Tetra tank, making sure to align color-coded plugs and jacks.n.Insert electrical leads into power supply.o.Run gels at a power output of 80 V on a magnetic mixer with continuous stirring.
Alternative: Our laboratory uses a magnetic stirrer from Thermo Fisher Scientific, but any similar instrument with sufficient surface on which to place the experimental setup will work.p. Turn off power supply and remove electrical leads when dye front reaches reference line at bottom of gels.Alternative: Our laboratory uses the ChemiDoc XRS+ System to visualize membranes, but any similar chemiluminescent imaging system and software will suffice.f.Use the straight-line tool to enclose peaks for each sample.g.Using the wand tool, click the inside of each peak to measure its area.h.Export the peak areas to statistical software (our laboratory uses GraphPad Prism) for further analysis.i. Normalize experimental samples (e.g., p-S6 and S6) to housekeeping protein (e.g., b-actin).j.Determine change in phosphorylation of S6 protein with lentiviral infection as: normalized p-S6/normalized S6.

EXPECTED OUTCOMES
The use of in vitro intestinal enteroids as a model for the in vivo intestine has become prevalent due to the translational opportunities provided by these tissues. 25Using source tissue derived from preterm infants, these cultures can be applied to studies of nutritional absorption and ontogenetic nutrient sensing within the intestine.Lentiviral knockdown of TSC2 serves as a model of mTORC1 overexpression, a state associated with epithelial differentiation 26 and increased intestinal regeneration post-injury. 27We opted for lentiviral gene knockdown over alternative methods because lentiviruses transfect both dividing and senescent cells, resulting in relatively high transduction efficiency. 28Here, we provide images of optimal (Figure 1A) and suboptimal (Figure 1B) enteroid morphologies for transfection.In addition, we provide examples of experimental readouts to evaluate lentiviral transduction efficiency and explore the effects of lentiviral gene knockdown in enteroids.Figure 2B illustrates representative results of flow cytometry analysis of transduction efficiency and cell viability following lentiviral transfection, while Figure 2C highlights examples of western blots from transfected enteroid cell lysates.Quantification of western blot bands through densitometry is also demonstrated in Figure 2C.In addition to those techniques demonstrated here, qRT-PCR, enteroid growth assays, and ELISAs, for example, represent further downstream assays to evaluate the effects of enteroid TSC2 knockdown.

LIMITATIONS
This protocol utilizes donor tissues derived from intestinal surgical resections.Due to donor variability, enteroid phenotypes may differ in response to both lentiviral transfection and the resultant gene knockdown.During enteroid maintenance, our laboratory routinely uses antimicrobial agents to inhibit microbial and fungal contamination.However, we discontinue use of these compounds during experiments as they are known to alter cellular physiology. 29Therefore, investigators should be cognizant of increased risk for microbial contamination during experimental periods.As with all enteroid models, the cell types represented in these 'mini intestines' are only of epithelial lineage.Because these models exclude mucosal immune cells, nerves, muscle, and a functional microbiome, enteroids cannot truly be considered representative of in vivo physiology.Finally, a plethora of enteroid media variations have been described.As use of different media recipes will likely alter experimental results, enteroid media composition should be seriously considered when evaluating experimental variability.

TROUBLESHOOTING Problem 1
Matrigel did not completely coat bottom of well, related to Preparation Step 1.

Potential solution
Ensure Matrigel and the plate in which Matrigel is deposited remain on ice during the entirety of the plating procedure.During Matrigel plating, aspirate ice-cold PBS from only one well at a time to ensure plate does not warm or dry during procedure.

Problem 2
Enteroids do not dissociate well into a single-cell suspension, related to Preparation Step 2.

Potential solution
Leave enteroids in TrypLE Express in 37 C incubator for slightly longer.Monitor cells often to ensure cell viability does not suffer.After transferring well contents to a 15 mL centrifuge tube and mechanically disrupting enteroids 20-30 times with a pipette, pass cells through a 37 mm cell strainer (e.g., STEMCELL Technologies, 27215) to eliminate cell clumps.

Problem 3
Cell viability is low, related to Preparation Step 3.

Potential solution
Perform manual (hemocytometer) counting to verify numbers from automated cell counter (e.g., Countess 3).Reduce incubation time in TrypLE Express.Ensure dead, unattached cells are removed via media change the day after plating, before completing second layer of Matrigel sandwich.Include 2.50 mM CHIR-99021, a GSK-3b inhibitor, 30 in culture media when preparing single-cell suspension in order to increase viability of stem cells, in particular. 31

Problem 6
Cell concentration is low for flow cytometry analysis, related to Step 5.

Potential solution
Cells stick to polystyrene tubes, so ensure polypropylene 5 mL tubes are used.Increase concentration of BSA in cell diluent when running flow cytometry to avoid cells sticking to tube walls.Include more wells/treatment to increase cell density.During all steps involving cell transfer, pipette tips and tubes can be pre-treated with 5% BSA in PBS to inhibit cell adherence to plasticware.

Problem 7
Flow cytometry compensation is difficult when using fluorescent protein-expressing samples, related to Step 5.

Potential solution
Use either GFP compensation beads (matching the specific GFP expressed by lentiviral particles) or constitutively GFP-expressing cell line to standardize compensation of GFP expression generated by enteroid transfection.

Problem 8
The top of the PVDF membrane sticks to the transfer gel, related to Step 10.

Potential solution
Saturate PVDF membranes for a longer period in transfer buffer to ensure membranes do not stick to the gel and the transfer of high molecular weight proteins of interest, in particular, is not disrupted.

Figure 1 .
Figure 1.Representative bright-field photomicrographs of human preterm infant ileal enteroids Scale bars represent 200 mm.(A) Enteroids characterized largely by spheroid (stem-like) morphology, optimal for lentiviral transfection and puromycin selection.(B) Highly differentiated enteroids (budding in center of image), unlikely to result in sufficient transduction efficiency.(C) Enteroid single-cell suspension, several hours post-plating.Note single-cell morphology, but clumps of individual cells forming.
t.Following incubation, pipette 100 mL of WRNE onto each Matrigel sandwich.CRITICAL: Add 100 mL PBS to unused wells to avoid evaporation in experimental wells.Note: Transfected cells should express green fluorescent protein (GFP) within 36-48 h posttransfection.u.Replace media with fresh WRNE every other day.v.A minimum of 4 days post-transfection, collect cells for flow cytometry measurement of transduction efficiency (Steps 2-5).

2 .
Prepare viability dye and cell diluent.a. From the LIVE/DEAD Fixable Red Dead Cell Stain Kit, allow component A (red fluorescent reactive dye) and component B (anhydrous DMSO), stored at À80 C, to reach 20 C-22 C. Note: Ensure DMSO is thawed before opening vials.b.Add 50 mL DMSO to vial of red reactive dye.Thoroughly mix contents and visually confirm complete dissolution of dye.

4 .
Cell viability staining (Troubleshooting).a. Centrifuge 5 mL polypropylene tubes at 400 3 g for 5 min at 4 C. b.Discard supernatant and resuspend cell pellet in 1 mL ice-cold PBS.Note: When using amino-reactive dye for staining, avoid resuspension/washing of cells in buffers containing extraneous proteins, such as BSA.c.Using a 2 mL pipette and 10 mL tips, add 1 mL reconstituted (Steps 2a-b) red reactive fluorescent dye to each 5 mL tube.Pipette up and down to mix well.d.Incubate tubes at 20 C-22 C for 30 min in the dark.e.Following 30 min incubation, centrifuge 5 mL tubes at 400 3 g and 4 C for 5 min to pellet cells.f.Remove supernatant and wash cells with 1 mL PBS.g.Repeat Step 4e.h.Discard supernatant and resuspend cell pellet in 1 mL 10% NBF.i. Incubate cell suspension at 20 C-22 C for 15 min in the dark.j.Centrifuge 5 mL tubes at 400 3 g and 4 C for 5 min to pellet cells.k.Discard supernatant and wash cells with 1 mL ice-cold PBS containing 1% BSA.l.Repeat Steps 4j-k.m.Place 5 mL tubes on ice.

Figure 2 .
Figure 2. Downstream analyses of transduction efficiency and gene knockdown effects (A) Schematic of lentiviral transfection of mammalian cell.(B) Representative flow cytometry scatterplot (left, 4 quadrants), GFP expression in non-target shRNA transfection: Q1-UL = dead cells not expressing GFP; Q1-UR = dead cells expressing GFP; Q1-LL = live cells, not expressing GFP; Q1-LR = live cells, expressing GFP.On right, a histogram depicting FITC intensity along X-axis and number of events at that intensity on the Y-axis.(C) Representative western blot images for control (non-target shRNA transfection) and TSC2 knockdown (20 MOI) in preterm human enteroids.S6, pS6, extracellular signal-regulated kinase 1/2 (ERK-1/2), and p-ERK-1/2 are shown normalized to b-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), respectively.Normalized phosphorylation S6 and ERK-1/2 is quantified in bar graphs.Enteroid TSC2 knockdown indicates an increase in phosphorylation of the ribosomal S6 protein, but no change was observed in the phosphorylation of ERK-1/2, an upstream target of mTOR.

16 .
Densitometry normalization and quantification of bands using ImageJ.a.To quantify signal emitted by a protein band, export membrane image from the ChemiDoc XRS+ System as a TIFF file.b.Open TIFF file in ImageJ.c. Use the rectangle tool to define the region of interest (ROI) around first lane.d.Press 1 to select rectangle box as first sample.Drag box to second sample and press 2 to select box as second sample.e. Press 3 to generate profile plot for ROIs.
When working with lentiviruses, care should be taken to avoid surface contamination.Decontaminate equipment and disposable supplies, both liquid and solid, with 20 min of 10% bleach or a similar oxidizing or decontaminating agent dictated via institutional/country regulations.Coat one 96-well microplate with Matrigel, as per Preparation Step 1. c. Dissociate enteroids into a single-cell suspension, as per Preparation Step 2. d.Count cells, as per Preparation Step 3. e. Centrifuge tubes at 400 3 g at 4 C for 5 min to pellet cells.f.Remove supernatant.Calculate the total volume of transduction media required to resuspend cells such that 100 mL of final cell suspension will seed each well of the 96-well plate with an empirically determined cell density (for our laboratory, 6 3 10 4 cells/mL, or a cell number sufficient to coat approximately 75% of each well). .Plate 100 mL/well of cell suspension in each well of the remaining, uncoated 96-well plate.Refer to Preparation Step 6 to calculate number of lentiviral particles needed per well.
ProtocolNote:Note: Cell pelleting (as in Step 2g) may be required if tube media already exceeds target volume.g.Add MISSION lentiviral transduction particles at an MOI of 20 (or alternative, as optimized in Preparation Step 6) to transfect cells.h 3. Dissociate enteroids into single-cell suspension.a. Warm TrypLE Express in a 37 C water or bead bath.b.Aspirate media from each well of a 96-well plate of transfected enteroids.c.Add 200 mL prewarmed TrypLE Express to each well.d.Mechanically disrupt Matrigel sandwiches in each well 10 times and place plate in 37 C incubator for 10 min.e.Following incubation, add 100 mL ice-cold PBS to dilute TrypLE.f.Pipette up and down 10-20 times to further dissociate enteroids.g.Transfer contents of one group of wells (defined by treatment group) into 5 mL polypropylene round-bottom tube.h.Repeat Step 3g for remaining groups.i. Centrifuge 5 mL polypropylene tubes at 400 3 g and 4 C for 5 min to pellet cells.j.Remove supernatant and replace with 1 mL PBS to wash cells.
6. Enteroid collection.a. Place a CoolSink XT 96F on ice a minimum of 30 min before enteroid harvest.b.Remove 96-well microplate containing enteroids from incubator.c.Place microplate on pre-cooled CoolSink XT 96F, and maintain on ice.d.Aspirate WRNE media from each experimental well of the 96-well microplate.e. Add 200 mL TrypLE Express to each well, and keep microplate on ice for 1 min.
10. Semi-wet protein transfer(Troubleshooting). Western transfer buffer should be prechilled to 4 C. a. Cut PVDF membrane to size of gel.b.In a large Petri dish, soak PVDF membrane in 100% methanol for 2 min.c.After methanol incubation, replace methanol with western transfer buffer for 15-20 min on a platform rocker.d.Similarly, soak filter paper and foam pads (from Mini Trans-Blot electrophoretic transfer cell) in western transfer buffer for 15-20 min.e.Remove tank lid from electrophoresis setup and lift out electrode assembly, pouring off and discarding running buffer within the inner buffer chamber.f.Open assembly arms and remove gel cassettes.g.Remove gels from gel cassettes by separating the two plates of the cassettes using provided spatula.h.Rinse gels briefly in water and, using a disposable Petri dish, equilibrate with 4 C western transfer buffer for 15 min.i.Place electrophoretic cassette, gray side down, on a flat, clean surface.j.Place one presoaked foam pad on the gray side of the cassette.k.Place filter paper sheet on foam pad.l.Carefully place equilibrated gel on top of filter paper.m.Place presoaked PVDF membrane on top of gel.n.Place a piece of filter paper on PVDF membrane.o.Complete the transfer sandwich by adding a second foam pad.p.Roll out any bubbles forming within the stack.q.Close the cassette without moving the transfer sandwich.r.Place cassette in module.s.Repeat for second gel, if applicable.t.Add frozen blue cooling unit.u.Fill the tank to the blotting mark with 4 C western transfer buffer.v.Add stir bar to tank, and place lid on unit.To remove the western transfer buffer from the PVDF membrane, wash the PVDF membrane in MilliQ H 2 O 3 times for 1 min each on a rocker.b.Immerse membrane in Ponceau S staining solution for 5-10 min on a rocker.Our laboratory uses a platform rocker from Corning, but any rocker with a sufficiently large, non-slip surface and variable speeds will suffice.Blocking in either scenario should occur on a rocker at 4 C for 1 h.13.Incubation with primary antibody.a.After blocking, wash the PVDF membrane with TBST a minimum of 2 times for 5 min each on a rocker.b.After washing, incubate PVDF membrane with primary antibody diluted in TBST with 3% BSA or 5% non-fat milk, per Step 12b.c.Incubate PVDF membrane in primary antibody for 10 h at 4 C with continuous rocking.After incubation with secondary antibody, wash PVDF membrane 3-4 times with TBST for 5 min each at 20 C-22 C on a rocker to remove any unbound secondary antibody.15.Substrate detection.a. Prepare working solution of equal parts SuperSignal West Pico PLUS Luminol/Enhancer Solution and Stable Peroxide Solution to cover surface of the membrane.b.Incubate PVDF membrane in working solution for 5 min at 20 C-22 C by gently shaking by hand to ensure surface of membrane is evenly covered.c.Remove PVDF membrane from working solution and place blot development folder over membrane.d.Image membrane using Image Lab software associated with the ChemiDoc XRS+ System.
Enteroid transduction efficiency is low, related to Step 1. MOI is suboptimal for vector/cell type.Evaluate transduction efficiency of higher MOIs using control, GFP-expressing particles.Lentiviral particles have degraded due to freeze/thaw cycles, thereby decreasing the functional titer.Always prepare lentiviral particles on ice and aliquot into small working volumes for storage at À80 C. Spinoculation speed was insufficient to ensure direct contact of enteroids/viral particles.Verify cells are localized at bottom of 96-well microplate after spinning.Puromycin concentration is ineffective.Redo puromycin trial and optimize for 100% cell death in uninfected cells.Cell fixation affects GFP fluorescence during flow cytometry because 10% NBF contains 4% formaldehyde, related to Step 4.