Protocol

Yeast Transformation Protocol (INVSc1 with pGBKT7)

YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose), YPD plates (YPD medium + 1.5-2% agar), SC-Trp plates (Synthetic Complete medium lacking tryptophan + 1.5-2% agar), 10× TE buffer (0.1 M Tris-HCl, 0.01 M EDTA, pH 8.0), 10× LiAc solution (1 M Lithium acetate, pH ~8), 50% PEG (w/v), sheared salmon sperm DNA (10 mg/mL), pGBKT7 plasmid DNA, sterile ddH₂O

2. Equipment

Cell culture incubator (30°C shaker), centrifuge, micropipettes, 1.5 mL and 50 mL centrifuge tubes, water bath (42°C), spectrophotometer

II. Experimental Procedures

1. Overnight Culture

Pick a single colony of wild-type INVSc1 from a YPD plate and inoculate it into a tube containing 3 mL of YPD liquid medium. Incubate overnight at 30°C with shaking at 160 rpm for 12 hours.

2. OD Measurement and Culture Expansion

Measure the OD₆₀₀ of the overnight culture using YPD medium as a blank and calculate the volume of culture required to achieve an OD₆₀₀ of 0.1 in 1 mL. Inoculate a flask containing 50 mL of YPD liquid medium with the calculated volume of overnight culture and incubate at 30°C with shaking at 160 rpm for approximately 4 hours until the culture reaches an OD₆₀₀ of approximately 0.5.

3. Plasmid Preparation and Cell Harvest

Resuspend the extracted pGBKT7 plasmid DNA in 40 μL of sterile ddH₂O. Transfer the yeast culture to a sterile 50 mL centrifuge tube and centrifuge at 4200 rpm for 2 minutes. Discard the supernatant, resuspend the cell pellet in 5 mL of sterile ddH₂O, then centrifuge at 4200 rpm for 2 minutes and discard the supernatant.

4. TE/LiAc Washes

Add 5 mL of freshly prepared TE/LiAc solution (1×) to the pellet and centrifuge at 4200 rpm for 2 minutes. Discard the supernatant and repeat the TE/LiAc wash step once more. Add 300-400 μL of TE/LiAc solution (1×) to the pellet, resuspend the yeast cells thoroughly, and transfer 0.1 mL of suspension into each 1.5 mL microcentrifuge tube.

5. Carrier DNA and Plasmid Addition

Add 10 μL of boiled and iced salmon sperm DNA and 10-20 μL of the pGBKT7 plasmid DNA to each tube. Incubate at 30°C with gentle shaking at 60 rpm for 30 minutes.

6. PEG/LiAc Addition and Heat Shock

Pre-heat a water bath to 42°C. After incubation, add 700 μL of the freshly prepared PEG/LiAc solution to each tube and mix gently. Incubate the tubes at 42°C for exactly 20 minutes.

7. Recovery and Plating

Immediately transfer the tubes to ice and incubate for 2 minutes. Centrifuge at 5000 rpm for 2 minutes and discard the supernatant. Add 1 mL of sterile ddH₂O to each tube, resuspend the pellet gently, centrifuge at 5000 rpm for 2 minutes, and discard the supernatant. Repeat the sterile ddH₂O wash step once more. Add 300 μL of sterile ddH₂O to each tube, resuspend the cell pellet thoroughly, and plate 100 μL of the resuspended cells onto SC-Trp agar plates. Incubate the plates at 30°C until single colonies appear (typically 2-4 days) and screen single colonies for transformants.

Yeast Colony PCR Validation Protocol

I. Materials and Equipment

1. Reagents

20 mM NaOH lysis solution (1 μL 1 M NaOH + 49 μL sterile H₂O, prepare freshly), 2× PCR master mix (Taq polymerase, dNTPs, reaction buffer included), 10 μM forward and reverse primer working solutions, sterile ultrapure water, 50× TAE buffer stock (242 g Tris base, 57.1 mL glacial acetic acid, 100 mL 0.5 M EDTA, pH 8.0), agarose, nucleic acid stain (e.g., GelRed)

2. Equipment

Thermal cycler, micropipettes, 0.2 mL PCR tubes, microwave, gel electrophoresis apparatus, UV transilluminator (365 nm), gel casting tray with 8-well comb

II. Experimental Procedures

1. Yeast Colony Lysis

Dispense 20 μL of freshly prepared 20 mM NaOH solution into sterile 0.2 mL PCR tubes, one tube per colony. With a sterile pipette tip, pick a fresh yeast single colony approximately 1-2 mm in diameter and use the tip to pipette up and down in the NaOH solution for 5 seconds to fully suspend the cells. Place the tubes in a thermal cycler and execute the following program: 95°C for 4 minutes, 4°C for 1 minute 30 seconds, and repeat this cycle four times. The resulting lysate is used directly as PCR template, and both primers and lysate should be kept on ice after lysis.

2. PCR Amplification

On ice, assemble a 25 μL PCR reaction mixture containing 12.5 μL 2× PCR master mix, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 1 μL lysate template, and 9.5 μL sterile ultrapure water. Set the thermal cycling conditions as follows: initial denaturation at 95°C for 3 minutes, followed by 32 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds (adjust according to primer Tm), and extension at 72°C for 1 minute (calculate at 1 kb per minute), then final extension at 72°C for 5 minutes, and hold indefinitely at 4°C.

3. Agarose Gel Electrophoresis Verification

Weigh 0.3 g agarose and add to 30 mL 50× TAE buffer, then heat in a microwave until completely dissolved and cool to approximately 60°C. Add 5 μL nucleic acid stain and mix gently, then pour the solution into a casting tray fitted with an 8-well comb and allow to solidify at room temperature for 25 minutes. Retrieve PCR products from the thermal cycler and load samples (7 μL of each) in the following order: DNA marker, positive control, negative control, and test samples. Run the gel at a constant 70 V for 25 minutes or until the bromophenol blue dye has migrated approximately two-thirds of the gel length. Illuminate the gel with a 365 nm UV transilluminator to visualize results. A positive result shows a distinct, sharp band at the expected size, while a negative result shows absence of a band or presence of primer dimers only (100-200 bp).

Preparation of Culture Media Protocol (YPD Medium and SC Medium)

I. Materials and Equipment

1. Reagents

Yeast extract, peptone, glucose, amino acid-free yeast nitrogen base (YNB), complete nutrient supplement mixture (SC powder), agar powder (for solid media), uracil, distilled water, individual amino acids (adenine, L-leucine, L-alanine, L-lysine, L-arginine, L-methionine, L-asparagine, aminobenzoic acid, L-aspartic acid, L-phenylalanine, L-cysteine, L-proline, L-glutamine, L-serine, L-glutamic acid, L-threonine, L-glycine, L-tryptophan, L-histidine, L-tyrosine, inositol, L-isoleucine, L-valine)

2. Equipment

Conical flasks, graduated cylinder, magnetic stirrer with stir bar, balance, mortar and pestle, high-pressure steam sterilizer, biosafety cabinet, alcohol lamp, petri dishes, sealing film, newspaper

II. Basic Formulas

YPD Medium (100 mL, liquid)

Yeast extract 1 g, peptone 2 g, glucose 2 g. After sterilization, add 50 μL of uracil to every 50 mL of YPD medium.

SC Medium (100 mL, liquid)

Glucose 2 g, amino acid-free yeast nitrogen base (YNB) 0.67 g, complete nutrient supplement mixture (SC powder) 0.2 g.

SC Powder (5 g per batch)

Adenine 0.05 g, L-leucine 1.0 g, L-alanine 0.2 g, L-lysine 0.2 g, L-arginine 0.2 g, L-methionine 0.2 g, L-asparagine 0.2 g, aminobenzoic acid 0.02 g, L-aspartic acid 0.2 g, L-phenylalanine 0.2 g, L-cysteine 0.2 g, L-proline 0.2 g, L-glutamine 0.2 g, L-serine 0.2 g, L-glutamic acid 0.2 g, L-threonine 0.2 g, L-glycine 0.2 g, L-tryptophan 0.2 g, L-histidine 0.2 g, L-tyrosine 0.2 g, inositol 0.2 g, uracil 0.2 g, L-isoleucine 0.2 g, L-valine 0.2 g.

When preparing dropout media, ensure the lacking amino acid is not added (e.g., do not add uracil for SC-Ura medium).

III. Experimental Procedures

1. Preparation of SC Powder

Clean a mortar with tap water, then rinse it with distilled water. According to the basic formula of SC powder, use a balance to weigh each amino acid powder and transfer them into the mortar, ensuring that when preparing dropout media, the lacking amino acid is not added. Gently grind the mixture with a pestle until the powders are fully mixed, then transfer the prepared SC powder into a clean empty bottle and label the bottle with the medium type (e.g., SC-Ura). Clean the mortar with tap water to remove any residual powder, rinse with distilled water, and store properly.

2. Preparation of Culture Media

Clean a conical flask with tap water, then rinse it with distilled water. According to the basic formula of the specific medium, weigh each component using a balance and transfer them into the conical flask. For solid media, add 2 g/100 mL agar powder to the liquid medium formula. Measure the required volume of distilled water using a graduated cylinder and pour it into the flask. Place a cleaned stir bar into the flask, set it on a magnetic stirrer, and stir until all solid powders are completely dissolved and no particles remain. Dispense the medium into smaller flasks in volumes of 50 mL or 100 mL per bottle, then seal the flasks with two layers of sealing film and newspaper. Sterilize them in a high-pressure steam sterilizer at 115°C for 20 minutes.

3. Post-Sterilization Storage

For liquid media, after sterilization, ensure the seals are intact and store the bottles at room temperature. For solid media, after sterilization, allow the medium to cool to 50-60°C, then pour plates inside a biosafety cabinet. Let the plates solidify overnight in the cabinet, then wrap them in newspaper and store in a refrigerator at -20°C.

4. Pouring Plates

Turn on the UV light of the biosafety cabinet 30 minutes in advance for sterilization. Sterilize the operator's clothing, hands, and utensils before placing them inside the cabinet, then ignite the alcohol lamp inside the cabinet. Place sterilized petri dishes near the flame, remove the seal from the conical flask containing the medium, and briefly pass the mouth of the flask through the flame. With your left hand, slightly lift the lid of the petri dish to create an opening wider than the flask's mouth, and with your right hand, pour approximately 10-20 mL of medium into the dish. Immediately cover the dish with the lid and gently swirl to distribute the medium evenly. Leave the plates in the biosafety cabinet to solidify overnight.

Screening, Microscopic Examination and Purification Culture of Yeast Transformants

I. Materials and Equipment

1. Reagents

Transforming yeast plate (SD selective medium), sterile PBS buffer

2. Equipment

Micropipettes, sterile pipette tips, centrifuge tubes (1.5 mL), slides and coverslips, microscope (40× and 100× oil immersion lenses), corresponding solid plates (for purification culture), sterile inoculation loop, marker pen

II. Experimental Procedures

1. Colony Picking and Slide Preparation

Use a marker pen to circle 5-10 colonies of different morphologies (size, color, degree of protrusion, etc.) at the bottom of the plate. With a pipette and sterile pipette tip or inoculation loop, gently pick up a small amount of colony material while avoiding the culture medium, then inoculate the colonies into 20 μL of sterile PBS that has been added to a 1.5 mL centrifuge tube or directly onto a slide. Gently pipette or shake to ensure the cells are evenly suspended while avoiding air bubbles. Drop 5 μL of the cell suspension onto a slide, cover it with a coverslip while avoiding air bubbles, and observe with 40× and 100× oil immersion lenses. Yeast cells appear oval or round with budding reproduction (small buds can be seen), while contaminating bacteria are smaller, rod-shaped or spherical without budding. Take photos or draw pictures and mark the colony numbers of suspected yeast.

2. Plate Zoning and Purification Culture

Use a marker pen to draw four quadrants (or more, depending on the number of colonies) at the bottom of the YPD plate and mark the colony numbers corresponding to each quadrant (such as "#1, #2..."). For each quadrant, use a pipette with a sterile pipette tip or an inoculation loop to perform streak plating for purification. Place the plate upside down at 30°C and culture for 2-3 days until single colonies grow.

Hydrogen Peroxide Treatment for Validation of Plasmid pGBKT7-pTRR1-EGFP

I. Materials and Equipment

1. Reagents

SC-Trp liquid medium (Synthetic Complete medium without tryptophan: 2% glucose, 0.67% YNB, 0.2% SC powder), 30% hydrogen peroxide stock solution, 1M hydrogen peroxide working solution (prepare freshly: 102 µL 30% H₂O₂ + 898 µL sterile water), sterile water

2. Equipment

Shaker incubator (30°C), spectrophotometer, centrifuge, micropipettes, 1.5 mL centrifuge tubes, sterile 12-well plates, sterile 96-well plates, flow cytometer, microplate reader

II. Experimental Procedures

1. Yeast Culture

Pick a single colony of the plasmid pGBKT7-pTRR1-EGFP transformant from an SC-Trp plate and inoculate it into 4 mL SC-Trp liquid medium. Incubate the culture in a shaker at 30°C and 220 rpm overnight for approximately 16 hours. Measure the OD of the overnight culture using SC-Trp liquid medium as a blank reference and calculate the volume N of the original culture needed to prepare 1 mL of culture with an OD value of 0.1. Add 50×N volume of the culture to a flask containing 50 mL of SC-Trp liquid medium and incubate the flask in a shaker at 30°C and 220 rpm for approximately 3.5 hours until the culture reaches the logarithmic growth phase with an OD value around 0.5.

2. Dose-Response Experiment 1 (Flow Cytometry Version)

Prepare 1 mL fresh 1M hydrogen peroxide working solution from the 30% stock. In 1.5 mL centrifuge tubes, prepare intermediate dilutions of hydrogen peroxide at different concentrations:

Target Final Concentration (mM)	1M H₂O₂ (µL)	SC-Trp Medium (µL)	Dilution Factor
4.0	4	996	250×
2.0	2	998	500×
1.5	1.5	998.5	667×
0.8	0.8	999.2	1250×
0.4	0.4	999.6	2500×
0.2	0.2	999.8	5000×
0(Control)	0	1000	—

Add 1 mL of logarithmic-phase yeast culture to each well of a sterile 12-well plate, pipette 10 µL from each intermediate dilution into the corresponding well (representing a 100× dilution to achieve the final target concentration), and mix gently. Incubate the 12-well plate in a constant-temperature incubator at 30°C for 1-1.5 hours. Transfer the sample from each well to 1.5 mL centrifuge tubes, centrifuge the samples at 4200 rpm for 3 minutes, discard the supernatant, and resuspend the pellet in 500 µL of sterile water. Use a flow cytometer to analyze EGFP fluorescence intensity in the corresponding samples.

3. Dose-Response Experiment 2 (Microplate Reader Version)

Prepare the intermediate hydrogen peroxide dilutions as described in the previous step. Add 200 µL of logarithmic-phase yeast culture to the corresponding wells of a sterile 96-well plate, pipette 2 µL from each intermediate dilution into the corresponding well (representing a 100× dilution to achieve the final target concentration), and mix gently. Incubate the 96-well plate in a constant-temperature incubator at 30°C for 1.5 hours, then use a microplate reader to analyze EGFP fluorescence intensity in the corresponding wells.

Flow Cytometry Experiments for Quantitative Analysis of EGFP Reporter Genes

I. Materials and Equipment

1. Reagents

Reporter strain (INVSc1 yeast strain carrying plasmid 13#), negative control strain (wild-type yeast strain without fluorescent protein), YPD+Uri medium, SC-Trp medium, 30% (w/w) hydrogen peroxide (H₂O₂) stock solution (analytical pure), phosphate buffered saline (PBS, pH 7.4, sterile, precooled at 4°C)

2. Equipment and Consumables

12-well plates, sterile 1.5 mL EP tubes, pipettes and sterile tips, constant temperature incubator (30°C), spectrophotometer (for OD₆₀₀ measurement), cryogenic high-speed centrifuge, flow cytometry instrument

II. Experimental Procedures

1. Yeast Culture and Stress Treatments

Reporter strains and wild-type strains were activated from plates, then cultured in 5 mL SC-T medium and 5 mL YPD+Uri medium overnight at 30°C with shaking. On the second day, the two overnight cultures were diluted into a new 50 mL SC-T medium to achieve a starting OD₆₀₀ of approximately 0.1. The culture was continued at 30°C in a shaking table for approximately 4 hours, and OD₆₀₀ was monitored regularly until it reached 0.4-0.6.

2. Preparation of Concentration Gradient

Prepare an intermediate diluent in 1.5 mL centrifuge tubes according to the following concentrations:

Intermediate diluent concentration (mM)	1M H₂O₂ (µL)	Medium SC-T (µL)	Target final concentration (µM)
0	0	1000µL medium YPD	0
0	0	1000	0
0.4	10µL 40mM H₂O₂	990	4
1	1	999	10
1.5	1.5	998.5	15
2	2	998	20
4	4	996	40
6	6	994	60
10	10	990	100
15	15	985	150
20	20	980	200
25	25	975	250
30	30	970	300
35	35	965	350
40	40	960	400

Prepare a 12-well plate according to the concentration gradient with three biological replicates per gradient, and add 990 µL bacterial solution to each well.

3. Preparation of H₂O₂ Working Solution (1M)

Dilute the 30% H₂O₂ stock solution with SC-T medium by taking 102 µL of the 30% H₂O₂ stock solution and adding 898 µL of SC-T medium, then mix well. Freshly prepare this working solution before each experiment.

4. Stress Treatment

Add 10 µL of H₂O₂ intermediate diluent to each well of the 12-well plate to achieve the final target concentration, with each group having three parallel controls. Put all the processing tubes back into the 30°C incubator and time for 60 minutes.

5. Flow Cytometry Analysis of Samples

After 60 minutes of processing, immediately take 1 mL of the culture from each processing well and transfer it to a labeled 1.5 mL centrifuge tube, then place the centrifuge tube on ice to stop all metabolic reactions within the cells. Centrifuge at 4200 rpm for 3-4 minutes at 4°C and collect the cell precipitate. Be careful to aspirate and discard the supernatant, add 1 mL of cold PBS, gently pipette to resuspend the cells, and centrifuge at 4200 rpm for 3-4 minutes. Be careful to aspirate and discard the supernatant, add 300-500 µL of cold PBS, and gently resuspend the cells by pipetting.

6. Flow Cytometer Setup and Data Acquisition

First, use the wild-type strain with 0 mM H₂O₂ sample for instrument setup. On the FSC vs. SSC scatter plot, adjust the voltages of FSC and SSC to clearly display the yeast cell population in the center of the graph. On the FITC vs. PE-Texas Red scatter plot, adjust the voltage of the FITC and PE-Texas Red channels so that the peak fluorescence signal of this "double-negative" cell population is at the lower end of the logarithmic coordinate axis (approximately 10³), leaving sufficient detection space for the positive signal. Collect signals from at least 10,000 cells for each sample. Run the samples in order from low concentration to high concentration, first running all the wild-type control groups, and then running all the experimental groups of the reported strains.

7. Quantitative Data Analysis

Gate P1 (Yeast Cells) on the FSC-H vs. SSC-H scatter plot by circling the yeast principal cell population with uniform morphology to eliminate cell debris and impurities. Gate P2 (positive cells) by displaying the single cells in Gate P1 on the Green-B map, and based on the signal of the wild-type blank control group, set a FITC positive threshold and circle all FITC-positive cells. For each sample, within the Live Cells gate, extract the Median Fluorescence Intensity (MFI) of the green fluorescence channel (FITC).

8. Expected Results

Under low-concentration H₂O₂ treatment, the proportion of FITC positive cells is relatively low, and as the concentration increases, the proportion of FITC positive cells significantly increases. The "corrected MFI" value should increase in a dose-dependent manner with the increase of H₂O₂ concentration and may reach a plateau at a certain concentration or start to decline due to cytotoxicity.

Culturing Saccharomyces boulardii in Simulated Intestinal Environment and Determining 24-Hour Growth Curve

I. Materials and Equipment

Reagents

Saccharomyces boulardii glycerol stock, YPD solid medium, simulated intestinal medium (glucose 4.4 g/L, arabinose 0.6 g/L, pectin 2.0 g/L, yeast extract 3.0 g/L, peptone 1.0 g/L, L-cysteine hydrochloride 0.5 g/L, 5% concentrated solution contains sodium bicarbonate 50 g/L, sodium dihydrogen phosphate 10 g/L, dipotassium hydrogen phosphate 10 g/L, magnesium sulfate heptahydrate 0.9 g/L, manganese chloride tetrahydrate 0.5 g/L, calcium chloride dihydrate 0.9 g/L, iron sulfate heptahydrate 0.05 g/L, zinc sulfate heptahydrate 0.05 g/L)

Equipment

Thermostatic orbital shaker, spectrophotometer or microplate reader, ultra-clean bench, incubator or water bath, Erlenmeyer flasks (250 mL), sterile pipettes and tips, cuvettes, anaerobic sealing bags with gas-generating sachets, sealing film

II. Experimental Procedure

1. Medium Preparation

Weigh reagents according to the above formula and dissolve in deionized water, adjust volume to 200 mL with thorough stirring until completely dissolved, applying gentle heating if necessary. Distribute 100 mL volumes into 250 mL Erlenmeyer flasks, seal flask openings with sealing film, and autoclave at 121°C for 20 minutes. After sterilization, cool the medium to room temperature or 37°C for use.

2. Inoculation and Anaerobic Treatment

In an ultra-clean bench, inoculate S. boulardii stock onto YPD medium and culture for 2 days for revival. Transfer single colonies of S. boulardii grown on YPD medium to test tubes containing 3 mL of specialized liquid medium for expansion culture, and after 4 hours inoculate into conical flasks containing 100 mL of specialized liquid medium. Immediately place the inoculated Erlenmeyer flasks into anaerobic sealing bags and transfer the entire sealed bag to the preheated shaker at 37°C.

3. Culture and OD Monitoring

Set shaker parameters to 37°C and 160 rpm for 24-hour cultivation. During the 24-hour cultivation period, sample at specific time points (0, 2, 4, 6, 8, 12, 16, 20, 24 hours) to measure OD values. For sampling, briefly pause the shaker and quickly remove the Erlenmeyer flask from the sealed bag, perform aseptic operations on the ultra-clean bench using sterile pipettes to withdraw 1 mL of bacterial suspension, immediately return the flask to the anaerobic bag, reseal, and promptly return to the shaker. Appropriately dilute the withdrawn samples using fresh medium to ensure OD values fall within the linear range of the spectrophotometer (0.1-0.8), measure the OD value of diluted samples at 600 nm wavelength and record, multiplying by the dilution factor to obtain the original bacterial suspension OD value.

4. Data Analysis

Plot the growth curve of S. boulardii under simulated intestinal conditions using cultivation time as the x-axis and OD₆₀₀ values as the y-axis, and analyze the lag phase, logarithmic growth phase, stationary phase, and death phase based on the curve.

Analysis of EGFP Gene Expression in TMA-Treated Yeast using Flow Cytometry and Fluorescence Microscopy

I. Materials and Equipment

Reagents

INVSc1 yeast strain harboring pRS416-TAAR5-GPA1-EGFP plasmid (positive transformant), wild-type INVSc1 yeast strain (negative control), YPD+Ura medium for wild-type INVSc1 yeast cultivation, SC-Ura medium (SC-U) for transformant cultivation, 1M TMA solution (4.78 g trimethylamine hydrochloride dissolved in ddH₂O to final volume of 50 mL), phosphate buffered saline PBS (pH 7.4, sterilized and pre-chilled to 4°C)

Equipment

Incubator shaker at 30°C, high-speed refrigerated centrifuge, BIO-RAD spectrophotometer, flow cytometer, fluorescence microscope with 100× oil immersion objective, 1.5 mL and 50 mL centrifuge tubes, micropipettes and sterile tips

II. Experimental Procedure

1. Strain Preparation and Pre-culture

Pick single colonies of pRS416-TAAR5-GPA1-EGFP transformant and inoculate into 5 mL SC-U medium, pick single colonies of wild-type (WT) strain and inoculate into 5 mL YPD+Ura medium, and incubate both cultures overnight for approximately 16 hours in an incubator shaker at 30°C and 220 rpm.

2. Culture Expansion

The next day, dilute each overnight culture into 50 mL of respective medium (YPD+Ura for WT strain and SC-U for transformant) to achieve starting OD₆₀₀ of 0.1. Incubate cultures in incubator shaker at 30°C for approximately 4 hours while monitoring OD₆₀₀ regularly, and proceed to next step when culture OD₆₀₀ reaches 0.4-0.6 (mid-logarithmic phase).

3. TMA Treatment Preparation

Prepare TMA concentration gradients in 1.5 mL centrifuge tubes according to the table below, preparing three new 1.5 mL microcentrifuge tubes as replicates for each TMA concentration. Add 10 μL yeast culture to each tube followed by corresponding concentration of TMA intermediate solution to achieve final required concentration.

Table 1: Preparation of TMA Concentration Gradients

1 M TMA (µL)	SC-U Yeast culture	Final Target Concentration (mM)
0	YPD Yeast culture 1000	0
0	1000	0
1	999	1
5	995	5
10	990	10
50	950	50
100	900	100

4. Incubation and Flow Cytometry Sample Preparation

Incubate tubes containing final cultures in incubator shaker at 30°C and 160 rpm for 6 hours. Centrifuge tubes at 4200 rpm for 3 minutes at 4°C and carefully aspirate and discard supernatant. Wash cell pellet with 1 mL ice-cold PBS and resuspend cells thoroughly by pipetting or gentle vortexing. Centrifuge again at 4000 rpm for 3 minutes at 4°C and carefully aspirate and discard supernatant. Resuspend final cell pellet in 1 mL ice-cold PBS, and samples are now ready for flow cytometric analysis.

5. Flow Cytometer Setup and Data Acquisition

It is recommended to run all wild-type (WT) control samples first followed by all transformant experimental samples, proceeding from low to high concentration. Using WT sample + 0 mM TMA sample for voltage adjustment, adjust FSC and SSC voltages on FSC vs. SSC plot to position yeast cell population clearly within center of plot, and adjust FITC and PE-Texas Red channel voltages on FITC (GFP) vs. PE-Texas Red plot so that peak fluorescence signal of autofluorescence population is at low end of logarithmic scale (approximately 10³), ensuring sufficient dynamic range for detecting positive signals. Collect data for minimum of 10,000 cellular events per sample.

On FSC-H vs. SSC-H dot plot, draw gate P1 (live yeast cells) around morphologically homogeneous population of primary yeast cells to exclude debris and aggregates. Display single cells from gate P1 on green fluorescence (FITC) histogram, set fluorescence threshold based on WT control sample to define positive population, and gate all FITC-positive cells (gate P2). Record median fluorescence intensity (MFI) of green fluorescence channel (FITC) from cells within gate P1 (live yeast cells) for each sample.

6. Fluorescence Microscopy for EGFP Detection

Pipette 5 µL of experimental culture (TMA-treated) onto clean glass slide and place coverslip over it to create wet mount, repeating for untreated transformant control culture. Apply drop of immersion oil directly onto coverslip and carefully engage 100× objective lens with oil drop. Using bright-field illumination, locate field of view with suitable density of yeast cells, switch to 488 nm (FITC/GFP) fluorescence channel to visualize green fluorescence, and capture representative images for both experimental and control samples.

Cellular Biology Protocols

Investigation of Sodium Butyrate (NaB) Cytotoxicity on BV2 Cells

I. Materials and Equipment

Cell lines and reagents

BV2 cell line (CL-0493) and corresponding specialized culture medium (CM-0493A) bought from Procell (Wuhan, China), sodium butyrate (NaB), trypsin, PBS buffer

Equipment

Cell culture incubator (37°C, 5% CO₂), flow cytometer, centrifuge, micropipettes, 96-well plate, 15 mL centrifuge tubes

II. Experimental Procedure

1. Cell Culture and Seeding

Transfer the culture medium from BV2 passage cultures to a 15 mL centrifuge tube. Rinse cells with 1 mL PBS, then add 2 mL trypsin and incubate for 2 minutes. Stop trypsinization by adding 6 mL culture medium and transfer the cell suspension to a fresh 15 mL centrifuge tube.

Centrifuge cells at 1200 rpm for 3 minutes and discard the supernatant. Resuspend cells in 2 mL culture medium and seed into 96-well plates at 5000 cells per well in 18 replicates (6 groups × 3 wells per group). Incubate plates at 37°C with 5% CO₂ for 24 hours to achieve a cell confluence of around 60-70%.

2. NaB Treatment

Remove the 96-well plate from the incubator and aspirate culture medium. Add 100 μL of NaB-containing medium to five experimental groups at concentrations of 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, and 2 mM (three wells per concentration). Leave the remaining group untreated as a negative control. Return plates to the incubator for continued incubation, ensuring adequate NaB-cell contact.

3. Cell Viability Assessment Using CCK-8 Assay

A cell counting kit-8 was employed to evaluate the cytotoxicity of NaB. Following incubation, measure absorbance at 450 nm using a microplate reader to determine optical density (OD) values.

Investigation of Sodium Butyrate (NaB) Cytotoxicity on HT-22 Cells

I. Materials and Equipment

Cell lines and reagents

HT22 (Mouse hippocampal neurons) cells (CL-0697) and corresponding specialized culture medium (CM-0697) bought from Procell (Wuhan, China), sodium butyrate (NaB), trypsin, PBS buffer

Equipment

Cell culture incubator (37°C, 5% CO₂), flow cytometer, centrifuge, micropipettes, 96-well plate, 15 mL centrifuge tubes

II. Experimental Procedure

1. Cell Culture and Seeding

2. NaB Treatment

3. Cell Viability Assessment Using CCK-8 Assay

A cell counting kit-8 was employed to evaluate the cytotoxicity of NaB. Following incubation, measure absorbance at 450 nm using a microplate reader to determine optical density (OD) values.

Validation of NaB Neuroprotective Effects Against Alzheimer's Disease: BV2 Microglial Activation Model

I. Materials and Equipment

Cell lines and reagents

BV2 cell line (CL-0493) and corresponding specialized culture medium (CM-0493A) bought from Procell (Wuhan, China), sodium butyrate (NaB), lipopolysaccharide (LPS), reactive oxygen species (ROS) fluorescent probe, trypsin, PBS buffer

Equipment

Cell culture incubator (37°C, 5% CO₂), flow cytometer, centrifuge, micropipettes, 24-well plates, 15 mL centrifuge tubes, 1.5 mL microcentrifuge tubes

II. Experimental Procedure

1. Cell Culture and Seeding

Transfer the culture medium from BV2 passage cultures to a 15 mL centrifuge tube. Rinse cells with 1 mL PBS, then add 2 mL trypsin and incubate for 2 minutes. Stop trypsinization by adding 2 mL culture medium and transfer the cell suspension to a fresh 15 mL centrifuge tube.

Centrifuge cells at 1200 rpm for 3 minutes and discard the supernatant. Resuspend cells in 1.4 mL prepared DMEM medium and seed into 24-well plates at 10 μL per well to ensure uniform distribution. Incubate plates at 37°C with 5% CO₂ for 24 hours for cell adherence.

2. NaB Pretreatment of BV2 Cells

Remove the 24-well plate from the incubator and aspirate medium. Replace with 500 μL/well fresh DMEM medium. Add different volumes of 100 ng/mL NaB solution (0, 0.5, 1, 2.5, 5, 10, 25 μL) to create concentration gradients of 0, 0.1, 0.2, 0.5, 1, 2, and 5 mM NaB (three wells per concentration). Leave one group untreated as negative control. Return plates to the incubator for NaB preconditioning.

3. LPS Stimulation

Remove the 24-well plate from the incubator and aspirate medium. Replace with 500 μL/well fresh DMEM. Add 1 μL of 50 μg/mL LPS to each well, gently agitate plates for uniform LPS distribution, and return to the incubator. This treatment induces microglial inflammatory activation, mimicking the neuroinflammatory environment associated with Alzheimer's disease pathogenesis.

4. Reactive Oxygen Species (ROS) Detection

Aspirate the medium from the 24-well plate, wash cells with 1 mL PBS per well, and collect into 1.5 mL microcentrifuge tubes. Centrifuge at 1200 rpm for 3 minutes, discard supernatant and resuspend the cells with 90 μL PBS and perform fluorescence staining with DC-FHDA (diluted 2000 times). Incubate samples in darkness for 15 minutes, then centrifuge at 1200 rpm for 3 minutes, remove supernatant, and resuspend cells in 500 μL PBS. Perform flow cytometric analysis with appropriate fluorescence detection settings, analyzing 3000 cells per sample to assess cellular ROS levels following stimulation. Evaluating NaB's inhibitory effects on Alzheimer's disease-associated neuroinflammation and oxidative stress.

Saccharomyces cerevisiae Adhesion Assay with MCEC Cells (Quantitative)

I. Materials and Equipment

Cell lines and reagents

MCEC (Mouse Colonic Epithelial Cells), genetically modified Saccharomyces cerevisiae expressing adhesion proteins (Syn strain), DMEM/F-12 culture medium (50 mL DMEM/F-12 + 5 mL FBS + 500 μL penicillin/streptomycin), YPD+uri culture medium, trypsin, PBS buffer, yeast fluorescent dye C-DCFDA

Equipment

Cell culture incubator (37°C, 5% CO₂), flow cytometer, centrifuge, micropipettes, 24-well plates, 15 mL centrifuge tubes, 1.5 mL microcentrifuge tubes

II. Experimental Procedure

1. Cell Culture and Seeding

Transfer the culture medium from MCEC passage cultures to a 15 mL centrifuge tube. Rinse cells with 1 mL PBS, then add 2 mL trypsin and incubate for 2 minutes. Stop trypsinization by adding 2 mL culture medium and transfer the cell suspension to a fresh 15 mL centrifuge tube.

Centrifuge cells at 1200 rpm for 3 minutes and discard the supernatant. Resuspend cells in 2.5 mL prepared DMEM/F-12 medium and seed into 24-well plates at 100 μL per well containing 400 μL medium per well. Incubate plates at 37°C with 5% CO₂ for 24 hours to ensure cell adherence.

2. Yeast Culture Preparation (1 day prior)

Pick single colonies of Syn Saccharomyces cerevisiae from agar plates and inoculate into 3 mL sterile YPD+uracil liquid medium in sterile tubes prepared under aseptic conditions. Incubate cultures overnight at 30°C with 160 rpm orbital shaking.

3. Yeast Culture OD Measurement and Dilution

Measure overnight yeast cultures for OD₆₀₀ using YPD+uracil medium as blank control. Calculate and inoculate required volumes of overnight culture into 50 mL fresh YPD+uracil medium to achieve initial OD₆₀₀ ≈ 0.1. Incubate cultures at 30°C with 160 rpm shaking for approximately 4 hours until OD₆₀₀ reaches ~0.5.

4. Serial Dilution and Yeast Fluorescent Labeling

When OD₆₀₀ reaches ~0.5, prepare serial dilutions using 900 μL PBS + 100 μL yeast culture to achieve multiplicity of infection (MOI) ratios of 0, 0.1, 1, 10, 100, and 1000 relative to MCEC cell density, creating 6 dilution tubes. Add fluorescent yeast dye C-DCFDA to each tube and incubate in darkness for 20 minutes for labeling.

5. Co-culture Incubation

Prepare 10 mL antibiotic-free DMEM/F-12 medium (9 mL DMEM/F-12 + 1 mL FBS). Remove MCEC plates from the incubator, aspirate medium, and add 500 μL fresh antibiotic-free medium per well. Add fluorescently-labeled yeast dilutions at 100 μL per well (three wells per MOI group, 18 wells total). Incubate plates at 30°C for 1 hour.

6. Flow Cytometric Analysis

Remove plates from incubator and aspirate culture medium to remove non-adherent yeast. Wash cells with 500 μL PBS per well and collect into 1.5 mL microcentrifuge tubes. Centrifuge at 1200 rpm for 3 minutes, discard supernatant and resuspend cells in 500 μL PBS. Perform flow cytometric analysis with appropriate fluorescence detection parameters, analyzing 5000 cells per sample. This quantifies yeast adhesion by measuring fluorescence intensity of yeast-cell complexes, thereby determining the adhesion efficiency of adhesion protein-expressing Saccharomyces cerevisiae.

Saccharomyces cerevisiae Adhesion Assay with MCEC Cells (Qualitative)

I. Materials and Equipment

Cell lines and reagents

Equipment

Cell culture incubator (37°C, 5% CO₂), flow cytometer, centrifuge, micropipettes, 24-well plates, 15 mL centrifuge tubes, 1.5 mL microcentrifuge tubes

II. Experimental Procedure

1. Cell Culture and Seeding

Centrifuge cells at 1200 rpm for 3 minutes and discard the supernatant. Resuspend cells in 2.5 mL prepared DMEM/F-12 medium and seed into 35mm confocal culture dish at 100 μL per well containing 2 mL medium per well. Incubate plates at 37°C with 5% CO₂ for 24 hours to ensure cell adherence.

2. Yeast Culture Preparation (1 day prior)

3. Yeast Culture OD Measurement and Dilution

4. Co-culture Incubation

Remove MCEC cell dishes from the incubator, discard the culture medium, and replace with antibiotic-free medium. Then, according to the experimental design, add WT and Syn yeast cultures at different MOI gradients (0.1, 1, 10, 100) to the corresponding culture dishes and co-incubate in a 30°C incubator for 1 hour to promote yeast adhesion to MCEC cells.

5. Fluorescent Staining and Confocal Microscopy Detection

After incubation, discard the culture medium and wash once with PBS to remove non-adherent yeast. Add 1 mL PBS and yeast dye Alexa Fluor 594 Concanavalin A, then incubate in darkness for 1 hour for staining. After completing yeast staining, discard the culture medium again, wash once with PBS, then add DAPI dye to stain cell nuclei. After incubating in darkness for 15 minutes place the 35mm confocal culture dish directly under a confocal microscope for detection. By observing the fluorescence signals of yeast dye Alexa Fluor 594 Concanavalin A and DAPI, you can record and photograph the adhesion patterns under different MOI gradients and different yeast strains (WT/Syn).

Co-culture Experiment of BV-2 and HT-22

I. Materials and Equipment

Cell lines and reagents

HT22 (Mouse hippocampal neurons) cells (CL-0697) and corresponding specialized culture medium (CM-0697) bought from Procell (Wuhan, China), BV2 cell line (CL-0493) and corresponding specialized culture medium (CM-0493A) bought from Procell (Wuhan, China), fetal bovine serum (FBS), sodium butyrate (NaB), lipopolysaccharide (LPS), trypsin, PBS buffer, ROS fluorescent probe, APC anti-mouse TNF-α Antibody (Biolegend)

Equipment

Cell culture incubator (37°C, 5% CO₂), flow cytometer, centrifuge, micropipettes, culture dishes, 15 mL centrifuge tubes, Transwell inserts (with porous membrane), 24-well plates

II. Experimental Procedure

1. Cell Passage and Culture

Retrieve BV2 and HT22 cell cryopreserved stocks from -80°C storage and rapid melting at 37℃. Transfer the cell suspensions respectively to different 15mL centrifuge tubes containing 3mL of culture medium. Centrifuge at 1200 rpm for 3 minutes, discard supernatant and resuspend each cell type in 2 mL prepared DMEM medium. Add BV2 cell suspension to one culture dish containing 10 mL DMEM medium and HT22 cell suspension to another culture dish containing 10 mL DMEM medium. Incubate both dishes at 37°C with 5% CO₂ for 2-3 days to ensure cell adherence and growth.

2. Cell Suspension Preparation

For both of BV2 and HT-22 cells: Take the cells that have been subcultured 2 to 3 times, remove culture dish from incubator, aspirate medium, rinse with 1 mL PBS, add 2 mL trypsin and incubate for 2 minutes. Stop trypsinization by adding 2 mL serum-free DMEM medium. Transfer cell suspension to a 15 mL centrifuge tube.

Wash both cell types 2-3 times with PBS and resuspend in serum-free DMEM medium. Adjust cell density of both BV2 and HT22 cells to 5×10⁴-1×10⁵ cells/mL using a hemocytometer.

3. Transwell Co-culture System Setup

HT22 Cell Seeding: Seed HT22 cells in 24-well plates at appropriate density to serve as the lower chamber.

BV2 Cell Treatment: 100-200μL of BV2 cell suspension was added to the upper chamber of the Transwell chamber. BV2 cells were treated with different concentrations of NaB (0 mM, 1 mM, and 2 mM) for 24 hours. After replacing the supernatant with new medium, BV2 cells were treated with 100ng/mL LPS for 24 hours to simulate an inflammatory environment.

Co-culture Setup: Place Transwell inserts containing NaB-treated BV2 cells into wells containing HT22 cells. Incubate the entire co-culture system at 37°C with 5% CO₂ for 24 hours.

4. Flow Cytometric Analysis

Remove culture medium from both chambers. For BV2 cells: add 2 mL trypsin to upper chambers, incubate for 2 minutes, stop with 2 mL medium, and collect into 1.5 mL microcentrifuge tubes. For HT22 cells: add 2 mL trypsin to lower chambers, incubate for 2 minutes, stop with 2 mL medium, and collect into separate 1.5 mL microcentrifuge tubes.

Centrifuge both cell types at 1200 rpm for 3 minutes and discard supernatants. Resuspend cells in PBS and stain with ROS fluorescent probe and TNF-α antibody according to manufacturer's protocols for dual inflammatory marker detection. Incubate in darkness for the recommended time, then centrifuge cells at 1200 rpm for 3 minutes, discard supernatant, and resuspend cells in 500 μL PBS.

Perform flow cytometric analysis on both BV2 and HT22 cell populations to detect and analyze ROS and TNF-α inflammatory markers. Set up appropriate fluorescence compensation using unstained and single-stained controls. Analyze at least 10,000 cells per sample. This analysis evaluates NaB's anti-neuroinflammatory efficacy by comparing inflammatory marker expression in HT22 cells co-cultured with differently treated BV2 cells.