Functional assays with human patient-derived enteroid monolayers to assess the human gut barrier

Summary Here, we describe the use of polarized patient enteroid-derived monolayers (EDMs) to assess the impact of e-cigarettes on the human gut barrier. These EDMs can be adapted to culture in a 96-well plate for high-throughput screening. We model the effect of e-cigarettes by combining pathogens, enteroids, and e-cigarette vapor-infused media and assess gut barrier integrity, bacterial internalization, and inflammatory response of the gut epithelium. This protocol can be used to assess the effects of e-cigarette components on gut functions. For complete details on the use and execution of this protocol, please refer to Sharma et al. (2021).


SUMMARY
Here, we describe the use of polarized patient enteroid-derived monolayers (EDMs) to assess the impact of e-cigarettes on the human gut barrier. These EDMs can be adapted to culture in a 96-well plate for high-throughput screening. We model the effect of e-cigarettes by combining pathogens, enteroids, and e-cigarette vapor-infused media and assess gut barrier integrity, bacterial internalization, and inflammatory response of the gut epithelium. This protocol can be used to assess the effects of e-cigarette components on gut functions. For complete details on the use and execution of this protocol, please refer to Sharma et al. (2021).

BEFORE YOU BEGIN
Biopsies from healthy humans should be processed shortly after the sample collection. Biopsies should be preserved and transferred in wash media containing ROCK inhibitors on ice package. Crypt-derived stem cells were isolated from the colonic and ileal biopsies of healthy human subjects, grown as 3D organoids (Jung et al., 2011, Sato et al., 2009, Ghosh et al., 2020, Sayed et al., 2020a, Sayed et al., 2020c, Sharma et al., 2021. CRITICAL: It is recommended to isolate the stem cells from the biopsies at the same day of collection. Isolation of the stem cells was also successful if it is done within 48 h of samples collection as long as the biopsies are preserved in the same condition of sample collection (i.e same transport media and ice package).

Isolation of the stem cells from biopsies
Timing: 60-90 min 1. Digest colon or ileal tissues 2 mm in length using 1-2 mL of collagenase type I (2 mg/mL) containing gentamicin (50 mg/mL) dissolved in PBS at 37 C. Check the tissues every 10-15 min and do mechanical from L-WRN cells, ATCC CRL-3276 TM ) containing specific components such as Y27632 (ROCK inhibitor) and SB431542 (an inhibitor for TGF-b type I receptor), nicotinamide, A83-01 (an ACTIVIN /NODAL/TGF-b pathway inhibitor), Epidermal growth factor, and SB202190 (a p38 MAPK inhibitor) to support the growth and long-term cultures of 3D organoids (Jung et al., 2011, Sato et al., 2009, Lee et al., 2020. The concentration of the added inhibitors/ supplements is mentioned in Material and Equipment section. The media and supplements of human organoids were obtained from the HUMANOID CoRE (UC San Diego, CA, USA).

Growing and expansion of 3D organoids
Timing: 7-14 days 6. Culture the stem cells in Matrigel domes on low binding tissue culture plates in a 37 C 5% CO2 incubator 7. For the first passages (p0-p2), use the splitting ratio range (1:1) to (1:3) in 24-well low binding tissue culture plates. Make the Matrigel dome by spreading 15 mL of Matrigel and single cell mixture per well and incubate the plate upside down for 5 min in a 37 C 5% CO2 incubator. 8. One day after the splitting, the organoids are small in size ( Figure 1A), and the size and number of organoids increase with time by providing more fresh media containing the above mentioned supplements/inhibitors. When adequate amount of medium sized organoids (p0-p2) is developed in 24-well plate ( Figure 1B), expand the organoids within the Matrigel in more wells in 24-well plate with the ratio (1:1 to 1:3) depending on the organoid number and size. 9. Around passage 2 or 3 (p2-p3), expand the organoids from 24-well culture plate to 12-well culture plates. Split one to three wells of a 24-well to 1 to 3 wells of a 12 well low binding tissue culture plate depending on the organoid number and size. Spread 25mL of Matrigel and stem cell mixture per well and incubate upside down for 10 min in a 37 C 5% CO2 incubator (Jung et al., 2011, Sato et al., 2009). 10. After 6-7 days of growth, expand the organoids in 12-well low binding tissue culture plates using a (1:5) or (1:6) ratio when they become large sized ( Figure 1C). Healthy and adequate amount of medium and large sized 3D organoids are required to prepare 2D EDMs. 11. Check the organoids daily for continued growth and that the cells are healthy (Figures 1A-1C) and not dying (dead organoids appear black) ( Figures 1D and 1E). Change the organoid media with all supplements and inhibitors every 2-3 days.
CRITICAL: The growth of organoids, especially in the first few passages (after the isolation from the biopsies), requires special attention in determining the expansion (splitting) ratio. Using a high expansion ratio (for example 1:6 or 1:12) with poorly growing organoids in the first few passages (p0-p2) could lead to loss of the line.

MATERIALS AND EQUIPMENT Preparation of human monolayer EDMs
Note: The 50% CM media required for the preparation of human EDMs media should be without any inhibitors added.
The volume of human monolayer EDMs can be adjusted and the amount of ingredient added is modified accordingly.
The volume of human monolayer EDMs prepared should be determined according to the number of the transwells prepared. The EDM media should be fresh for each experiment.
Preparation of 50 mL media with cocktails for the culture of organoids (50 mL) Note: All the components are aliquoted and saved at -80 C till use. The volume of each aliquot/ component should be suitable for a single use to avoid freezing and thawing of the components.
CRITICAL: Repeated freezing and thawing of the aliquots should be avoided.

Note:
The preparation of all the added components (aliquots from each component are stored at -80 C for 2-3 months) mentioned in the table above is done as the following: Preparation of primary culture media (100 mL) (Stored at 4 C for 1-2 months according to the expire date)

Preparation of e-cigarette vapor-infused media
The mixture of e-cigarette liquid contains 70% propylene glycol, 30% glycerol without additives and/or flavors. The preparation of e-cigarette vapor infused media is done with or without 6 mg/ mL nicotine (Sharma et al., 2021, Alasmari et al., 2021, Alhaddad et al., 2020. E-cigarette vaporinfused media is generated by application of negative pressure on the activated battery, which causes the e-liquid to be heated and drawn through the internal atomizer and then a syringe including 10 mL of DMEM/F12 media containing 15 mM HEPES and 10% FBS. The infection media is developed by exposure to 50 mL of e-cig vapor generated from the vaporization of the e-cig liquid for 30 times, involving a 12-s interval between each exposure.

Preparation of LB broth (Stored at 4 C for 1 month)
Note: 20 g of LB broth includes 10 g of Tryptone, 5 g Yeast extract, and 5 g sodium chloride.
Autoclave the LB broth solution at 121 C for 20 min, and allowed to cool down to room temperature around 20 C-22 C . Make aliquots of the broth in 50 mL tubes.
Pause point: The LB broth aliquots can be kept at 4 C for 2-weeks-1 month.

Preparation of LB agar plate
Note: Autoclave the LB agar solution at 121 C for 20 min, and then allowed to cool down until the solution is warm, but does not solidify. Bacterial culture tri-plates are prepared by pouring 6 mL of LB agar solution per tri-section. Leave the plates at room temperature around 20 C-22 C until the agar solidifies.
Note: For the bacterial clearance assay, tri-plate is recommended over a single plate since it helps to test several bacterial dilutions from the same sample at the same time and same plate.
Pause point: The bacterial culture plates can be kept at 4 C for 2-4 weeks in the closed packet. At the time of use, the plate is kept at room temperature around 20 C-22 C for 10-15 min before adding the bacterial dilutions.

STEP-BY-STEP METHOD DETAILS Part 1: Development of human EDMs in 96-well inserts
Timing: 3-4 days Day 0 of the EDMs preparation 1. Prepare the Human monolayer EDM media required for the differentiation of the stem cells to colon specific cells. The EDM model resembles the physiologic gut lining in which all cell types (enterocytes, goblet, Paneth enteroendocrine, and tuft cells) are proportionately represented , Sato et al., 2009, Sayed et al., 2020a, Miyoshi and Stappenbeck, 2013). a. The components of the human monolayer EDM media is mentioned in the previous section under the title ''Materials and Equipment.'' b. Allow media to incubate at room temperature around 20 C-22 C during the step of EDMs preparation. Since the step of EDM preparation takes 30-45 min, the EDM media can be kept at room temperature around 20 C-22 C at the start of EDM preparation.
CRITICAL: The human monolayer media should be fresh, constituted on the day of EDMs preparation. The media can be kept at 4 C for few days (2-3 days). Storing the media at 4 C for a longer period could affect the degree of stem cell differentiation due to the stability and efficacy of the components added to human monolayer EDM media could be affected by long-term storage. 3. Splitting of 3D organoids into single cells a. Remove media from the organoid culture grown in 12-well plate, then add 1 mL of 0.5 mM PBS-EDTA to each well. b. Scrape cells/Matrigel in PBS-EDTA mixture and collect the cell suspension in a 15 mL conical tube, then centrifuge at 200 xg for 4 min to precipitate the cell pellets. No wash media is used in this step. c. Remove the supernatant above the pellets and add 300 mL of TrypLE / well to separate the cells from each other. The volume of TrypLE should be adjusted according to the numbers of wells collected i. Incubate the TrypLE suspension in a 37 C water bath for 5-15 min.

Prepare
ii. Disrupt the organoids mechanically in the TrypLE mixture by pipetting the cells up and down up to 20 times using a p1000 micropipette to have single or small clusters of cells.
Pipetting should be continued until no cell clumps/ aggreagtes are appeared. iii. Inactivate the TrypLE by adding a serum-containing buffer such as wash buffer at the ratio (1:5) (1 TrypLE: 5 washing media). iv. Centrifuge at 200 3 g for 4 min to remove the TrypLE and precipitate the dissociated cells.
CRITICAL: The time of TrypLE incubation depends on the density, size, and overall health of the organoids. Large and dense organoids require a longer incubation period with the TrypLE at 37 C. Inadequate incubation of organoids with the TrypLE or insufficient pipetting of the organoids could lead to incomplete dissociation of organoids into single stem cells. Alternatively, clumps of condensed cells are formed, which could affect the differentiation of the stem cells after that.

Filtration and counting the single cells,
a. Place a 70 mm filter over a 50 mL tube. Wet the filter with 200 mL human monolayer media (prepared at step 1). b. Resuspend the cells in the desired volume of human monolayer media to get a good concentration of cells and to allow complete resuspension of the cell pellet to avoid loss of cells in the filter ($200 mL per transwell). c. Filter the suspended cells using the pre-wet 70 mm filter over a 50 mL tube. d. Count the single cells or very small clusters of cells using the Trypan blue method or automated counter.
CRITICAL: The human monolayer media should be kept at room temperature around 20 C-22 C before the suspension of single cells.
CRITICAL: The volume of human monolayer media required to dissolve the single cells should be appropriate, 1 mL is a good start. If the concentration of cells is high, more media can be added till it reach the required concentration of cells.
CRITICAL: If the cells are more diluted in human monolayer media, the cells can be precipitated again by centrifugation at 200 3 g and then dissolved in the appropriate volume. 7. Change the media from the apical and basolateral sides of the transwells using the monolayer media. 8. Check the monolayer appearance under the microscope. After 24 h, you should see the cells start to flatten out, but all space between cells may not be filled until 48 h post-seeding. 9. Measure the transepithelial electrical resistance (TEER) using the REMS automated TEER machine (as will be discussed in detail in the next step). The TEER value after 24 h (first-time point) helps to follow the TEER kinetics of the monolayers at different time points, which indicates the monolayer's efficiency (Figure 2).
CRITICAL: The human monolayer media should be kept at around 20 C-22 C before changing the media and the media should be added smoothly and slowly on the apical side of the transwells (inserts) to avoid the detachment of the differentiated cells from the transwells.
Day 2 of the EDMs preparation (first exposure to vapor infused media) 10. Measure the TEER in the morning using the REMS automated TEER machine before applying any conditions. 11. Expose the basolateral side of the transwells to the e-cigarette vapor-infused media for 4 h. Single exposure of the EDMs with the vapor media for 4 h represents acute exposure) (Sharma et al., 2021). To mimic the vapor exposure to the gut, we expose the basolateral side of EDMs (which represents the gut epithelium side facing the blood), while the apical side of the EDMs represents the side of epithelium facing the internal GIT. 12. Collect supernatants from the basolateral side of the transwells at 4 h of exposure to assess the level of inflammatory cytokines. 13. Collect the supernatant from the apical part of the transwells, save the supernatant at -20 C until the analysis. The supernatant collected from apical and/or basolateral compartments can be used to assess the cytokine levels and other functional assays. 14. Collect the RNA lysate to assess the transcriptome change upon addition of e-cigarette vaporinfused media, Add RNA lysis buffer to the cells attached to the transwells, incubate the lysis buffer with the transwell for 2-3 min, then pipette the lysis buffer up and down 10 times and then collect the cell lysate in 1,5 Eppendorf tubes for further RNA extraction.
Pause point: The cell lysate in RNA lysis buffer is kept at -80 C till the step of RNA extraction. Supernatant collected can be kept at -20 C or -80 C till analysis for cytokine assay.

Day 3 of the EDMs preparation (chronic exposure for vapor infused media)
15. To assess the effect of chronic exposure of EDMs to e-cigarette vapor-infused media, expose the EDMs on Day 3 of the preparation continuously to e-cigarette vapor-infused media for 4 h, and the medium is refreshed with new e-cigarette vapor-infused media for repeated exposure (3 times exposure) prior to analyzing them at 24 h (Day 4 of EDMs preparation). Measure the TEER after 24 h for exposure. 16. Collect supernatants from the basolateral side of the transwells at 24 h of exposure to the vaporinfused media.to assess the level of inflammatory cytokines. 17. Collect the cell lysate for RNA extraction as described in the previous step.
Pause point: The cell lysate in RNA lysis buffer is kept at -80 C till the step of RNA extraction. Supernatant collected can be kept at -20 C or -80 C till analysis.
CRITICAL: In 96-well plate, the RNA extracted from one well is relatively low and insufficient to assess the transcriptome analysis. Therefore, 2-3 wells from each condition should be combined to get an adequate RNA concentration for qPCR and/or sequencing analysis. CRITICAL: After RNA extraction and measurement of the RNA concentration, aliquot the fresh RNA into 2 parts: 200-300 ng for RNA sequencing and 500 ng-1000 ng for transcriptome analysis by qRT-PCR. Repeated freezing and thawing for the extracted RNA should be avoided.
Note: The EDMs used in chronic exposure experiment (Day 3) are not the same ones used for acute exposure experiment (Day 2), but they are derived from the same organoid cultures.
Part 2: PC-controlled high-throughput automated TEER measurement for epithelial monolayer in 96-well plates (REMS AutoSampler) Timing: 5-60 min, depending on the numbers of wells to be measured 18. Place the plate on the REMS plate holder and ensure all 4 corners are properly placed and clipped in (Figure 2 and Methods video S1 CRITICAL: Calibration steps are important to ensure the electrodes will be correctly centered. Inappropriate calibration could lead to incorrect direction and movement of the Electrode, which could lead to the damage of expensive electrode. CRITICAL: The electrode returns home (the original position) after every run, and the A1 calibration steps should be run every time the electrode returns to home. The abrupt mechanical movement made when the probe returns home can cause a slight misalignment of the XY position.
Pause point: It is recommended to coat the unused wells with plastic adhesive sheets for future use without causing contamination of these wells in the current analysis.

Part 3: Gentamicin protection assay to assess bacterial internalization in the human EDMs
Timing: 4 days Day 0: Prepare the human monolayer EDMs as in protocol #1 in 24-well transwell (inserts) (Figure 3). 25. Follow the same steps in the human EDMs preparation as Part 1, starting from step 1 to step 4. 26. Plate 2 3 10 5 cells per well in 24-well corning transwell inserts.
a. The transwell inserts are coated with 130 uL of PBS-diluted Matrigel (1:40 dilution) with a dilution of 300 mg/mL. b. Add 700 mL of monolayer media to the basolateral side of the transwell. Add 200 mL of the cell suspension mix to the apical side of each well. c. The precautions and critical steps applied to the preparation of human EDMs in 96-well plate should also be applied in the preparation of EDMs in 24-well plate.
Day 1: 27. Change monolayer media on the apical and basolateral sides as described in protocol # 1 28. Culture E. coli LF82 in LB broth (Sayed et al., 2020a). We did the following steps : a. Make a day culture: inoculate one loopful of bacteria from an agar plate into 10 mL of LB in a 50 mL conical tube with a slightly loose lid to allow aeration. Incubate the culture at 37 C in an orbital shaker at 150 rpm for 6-8 h. b. Make overnight culture: Add 100 mL of the day culture to 10 mL of LB in a 15 mL conical tube.
Incubate at 37 C without shaking for overnight (about 16 h) and the lid should be tightly closed to allow minimum aeration and keep the bacteria invasive.
Day 2: Infection of EDM exposed to e-cigarettes with E.coli LF-82 29. Expose the EDMs to the e-cigarette vapor-infused media and E.coli inoculum.
Expose the basolateral side of the transwells to the e-cigarette vapor-infused media for 4 h (single exposure) or 3 times, each 4 h apart prior to analyzing them at 24 h (chronic exposure). CRITICAL: The infection media should be without antibiotics.
33. Kill the extracellular bacteria and lyse the cells for bacterial counting a. After 1-3 h of infection, add 200 mg/mL of gentamicin to kill the extracellular bacteria. b. After 90 min of incubation with gentamicin, remove the media from the wells used for the bacterial internalization at every time point. Add 150 mL of 1% Triton X-100 to the apical side of each transwell and incubate for 10 min in a 37 C 5% CO2 incubator. c. Lyse the cells by pipetting up and down 5-7 times. Do serial dilution of the cell lysate by adding 100 mL of lysate to 900 mL of 13 PBS (10 À1 dilution). Dilutions up to 10 À6 should be done depending on the cells.

EXPECTED OUTCOMES
Evaluation of the efficiency of the prepared EDMs in the 96-well plate: Following the steps of EDMs formation, Sharma et al. showed that the stem cells are differentiated into polarized enteroid derived monolayer with apical and basolateral sides that physiologically mimic the in vivo gut lining (Sharma et al., 2021). The EDMs include all intestine cell types (enterocytes, goblet, Paneth enteroendocrine, and tuft cells) that are proportionately represented as shown by other groups and us , Sato et al., 2009, Miyoshi and Stappenbeck, 2013, Noel et al., 2017, Ghosh et al., 2020, Sayed et al., 2020a, Sayed et al., 2020c, Sayed et al., 2020b. Several readouts indicate the differentiation of stem cells into polarized epithelial cells (a) TEER kinetic by REMS: the TEER value at day 1 (day of EDM preparation) is low (undifferentiated stem cells), after that the TEER value is increased gradually in day 2 and day 3 indicating the formation of tight junctions ll OPEN ACCESS STAR Protocols 2, 100680, September 17, 2021 among the differentiated epithelial cells. (b) Assessment of the differentiation and stemness transcripts by qRT-PCR: The transcript level of stemness marker (Lgr-5), and differentiation transcripts (carbonic anhydrase and sucrase isomaltase for enterocyte markers, chromogranin for enteroendocrine marker, b-defensin and lysozyme for Paneth cell marker, DCLK-1 for tuft cell marker, and Muc-2 for goblet cell marker) is measured in the differentiated EDMs and compared to undifferentiated stem cells. Compared to undifferentiated stem cells, the differentiated EDMs show a significant reduction in the stemness marker and a marked increase in the differentiation markers after 48-72 h of EDM preparation. (c) Immunofluorescence labeling of differentiation markers in the polarized EDMs. As the the EDMs are well differentiated, the expression of stemness markers (Lgr-5) is reduced in differentiated EDMs compared to undifferentiated EDMs. While the expression of differentiation markers (Villin, Carbonic anhydrase, MUC2) is increased in the differentiated EDMs. Moreover, the expression of tight junction proteins such as occludin and E-cadherin is increased in the differentiated EDMs Test the impact of e-cigarettes on the integrity of the gut barrier: E-cigarette disrupts the integrity of the human gut barrier and triggers inflammation (Sharma et al., 2021). We evaluated the impact of e-cigarette vapor exposure of the human EDMs after a single and repeated exposure. An acute single exposure to e-cigarettes was associated with a significant drop in TEER at 4 h, but the initial drop at 4 h was reversed after 24 h. Exposure of the EDMs to repetitive e-cigarettes exposures (3 times, resembles chronic exposure) caused a sustained drop in TEER at 24 h compared to control cells. There was a 3-fold increase in the percentage of burst tight junctions (TJs) TJs in the chronically exposed EDMs compared to control cells. Also, there was a significant drop in gene expression of ZO1 after chronic repetitive multiple exposures.
Effect of e-cigarettes on the bacterial internalization and inflammatory response of the gut epithelium Next, we assessed the impact of e-cigarettes exposure on gut reactivity to pathogens. Since e-cigarette disrupts the integrity of the human gut and increases leakiness (Sharma et al., 2021), we expected that the number of internalized bacteria is increased after repeated exposure to e-cigarette vapor. A single exposure to e-cigarettes vapor resulted in a slight increase in the number of internalized bacteria compared to control cells. While EDMs repeatedly exposed to e-cigarettes (33) showed a statistically significantly higher number of internalized bacteria compared with control EDMs after 3 h of infection, suggesting higher infectivity of gut epithelium after e-cigarette exposure. Also, the number of internalized bacteria was increased after repetitive exposure to e-cigarettes compared to a single exposure to e-cigarette vapor.

LIMITATIONS
Although the EDMs models mimic the in vivo physiological gut epithelium, one limitation of this model that it cannot be passaged and propagated. Since the 2D EDMs are derived from 3D organoids, there could be a batch-to-batch variation in the differentiated EDMs. Some components in the 3D organoid media such as Matrigel, fetal calf serum, growth factors, and inhibitors (mentioned in the table for preparation of 50% CM media) affect the proliferation of stem cells in the 3D organoids and hence could affect the differentiation of these cells into 2D EDMs especially after long maintenance of the stem cells on culture (Ji andWu, 2020, Drost andClevers, 2018). Therefore, the generation of 2D EDMs requires highly trained skills and regular quality controls. Furthermore, the abundance of differentiated cell types could slightly vary across independent biological replicates. Moreover, the development of 3D organoid and 2D EDMs are costly and require highly funding sources that are not matched with many research laboratories worldwide. Also, the EDM model mimics the gut epithelium, but coculture of the EDMs with multiple components such as immune cells, microbes, and vascular endothelial cells at the same time to mimic the gut environment is still challenging.

TROUBLESHOOTING Problem 1
Poorly Differentiated EDMs (steps 8-10) The prepared EDMs are poorly differentiated; this can be verified in the following conditions: (a) The TEER value is very low and has not changed after the EDMs preparation till 72 h post preparation. (b) The stemness marker and differentiation markers are comparable in the undifferentiated cells and EDMs. Although the measurement of TEER in 96-well plate is automatic, the preparation of EDMs is done manually.
Several causes could lead to poorly differentiated EDMS: (1) Unhealthy organoids from which the EDMs are generated; (2)An insufficient number of cells embedded in the transwell; (3) Old human monolayer EDMs media; (4) The wrong formulation of human monolayer EDMs media.

Potential solution
Solution 1: Generate EMDs from healthy organoids. Organoids with numerous black centers and disintegrated organoids are not good sources for differentiated EDMs. To reduce the development of unhealthy organoids: a) Regularly change the organoid media, especially when the media color change to yellow which is a marker for the accumulation of toxic metabolite and therefore the media should be changed. b) Passage/ Split the organoids when they reach confluency. Use the correct splitting ratio. Growing of the organoid should be done in 50% CM that favors the stemness. c) Do not use 50% CM media stored for at 4 C for more than a week. d) Be sure that 50% CM media include all the cocktail components as mentioned in the Materials and Equipment section.
Solution 4: Constitute the human monolayer EMDs correctly. Most of the components added to the 3D organoids are not required to the 2D EDMs, and their presence could affect the development of differentiated polarized EDMs.
Problem 2 E.coli LF82 did not infect the EDMs well (steps 29-32) The prepared EDMs are differentiated well, but the infection of EDMs with the bacteria did not lead to the expected findings. This problem can be raised due to the following reasons: (1)Incorrect moi used; (2) Use old bacteria culture; (3) Decrease the bacterial infectivity due to the temperature, and the OD.

Potential solution
Solution 1: Use the correct moi (1:30) to infect the EDMs, as the number of bacteria added depends on the number of cells added to the transwell.
Solution 2: The bacteria should be fresh, old bacteria do not replicate and be invasive enough in the EDM model. Also, the bacteria inoculum present in LB agar (source of infection) should not be older than 1 month. Confirm the bacteria growth on LB broth by measuring the OD600 of bacteria.
Solution 3: All infective steps should be performed with prewarmed material at 37 C. Optimal OD should be reached before infection (lower or higher OD influence the bacterial behavior and can affect the infectivity).

OPEN ACCESS
STAR Protocols 2, 100680, September 17, 2021 Problem 3 Variation in the colony-forming units (CFU) produced from different dilutions of the same samples (steps 33-34) The serial dilutions of the cell lysate containing bacteria (10 À1 to 10 À6 ) are done to assess the CFU. Some dilutions could give an inaccurate estimate of the actual bacterial cfu. This problem is probably due to errors in the preparation of bacterial serial dilutions.

Potential solution
Solution 1: Experiment should be performed in duplicate and repeated in case of incoherent results.
Solution 2: Serial dilutions should be performed with well calibrated pipettes, the solution should be homogenized and tips should be changed between each dilution steps.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Soumita Das (sodas@health.ucsd.edu)

Materials availability
EDMs generated in this study will be made available on request, but we may require payment and/or a completed Materials Transfer Agreement if there is potential for commercial application.

Data and code availability
This study did not generate/analyze a new dataset.