Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Experiments with human PCLS must be performed according to The Code of Ethics of the World Medical Association and approved by the local ethics committee (the exemplary PCLS experiments shown here were approved by the local ethics committee of the Hannover Medical School; number 2701-2015). Written informed consent to the use of human lung material is required from all donor patients or their next of kin. The results described in this manuscript were obtained with tissue slices from donors of different ages, genders, medical history, and cause of resection, though most of the tissue received was from patients suffering from lung cancer. Only tumor-free tissue was used for experiments.

1. General Preparation of Human PCLS and Subsequent Exposure to Chemicals

NOTE: Two persons are required to fill a lung. An up-to-date vaccination record for hepatitis A and B is recommended. Patients are routinely screened for HCV and HIV prior to lung transplantation. If an active infection with Mycobacterium tuberculosis is diagnosed or suspected, the lung should be rejected. Nevertheless, all fresh human lung tissue and samples derived from it must be treated as potentially infectious and corresponding protective measures must be taken (particle filter masks (FFP2), protective eyewear, gloves) to ensure occupational safety of the staff. The procedure takes 60 – 90 min.

1. Confirm the intactness of the human lung lobe.
   NOTE: A tear in the pleura prevents homogeneous filling of the tissue. Human lung material must be from the day of resection. Storage periods of about 2 h at room temperature (RT) prior to filling it with agarose on ice are tolerated. The tissue that we use in this protocol is obtained from patients undergoing resection. It is not from deceased patients. If the tissue has been stored on ice, pre-warm it to RT before filling it, otherwise the agarose will polymerize immediately, and no homogeneous filling will be possible.
   Caution: Ensure that all persons in contact with native human material put on protective clothing consisting of a lab coat, two pairs of gloves, cap, face mask, and a pair of safety glasses. Human material is potentially infectious.
2. Weigh 7.5 g of low-gelling agarose and add it to 250 mL of bi-distilled water. Boil the agarose in a microwave until the agarose is dissolved. Cool it to approximately 40 °C, depending on the melting and gelling point of the agarose.
   NOTE: Several flasks are required, depending on the lobe size.
3. Pre-warm and keep the culture medium (Table of Materials) at 37 °C.
   NOTE: If the agarose is too hot, vitamins in the culture medium will lose effectiveness and cells will be damaged. If the temperature of the medium/agarose solution is below 37 °C, the gelling process will start and impair homogeneous filling of the lung. Only a temperature range of 37 °C to 39 °C is recommended.
4. Place all the required materials within reach before starting: 5 – 10 clamps, flexible catheter of approximately 1 m length, a suitable syringe (e.g., 50 mL) fitting the connection to the catheter, and an ice box filled with ice.
5. Cannulate the trachea/main bronchus by inserting the silicone tube and fixating it with a clamp oriented parallel to the tube, so that the clamp squeezes the tissue together alongside the silicone tube without pinching it off. Verify that the silicone tube is fixated inside and cannot slip out during the filling procedure. Close all other bronchi, blood vessels and injuries with clamps, so that no agarose can leak out during the filling procedure.
6. Mix an equivalent volume of 3% low-gelling agarose with culture medium in a beaker. Instill the mixture into the lung using a 50-mL syringe. Prior to refilling the syringe with medium, clamp shut the catheter with fingers or a clamp to avoid air bubbles and agarose reflux. Fill the lung lobe until it is fully inflated. Carefully touch the lung pleura on the side; the pleura should be even and hard.
   NOTE: Depending on the size of the lung lobe, up to 2 or 3 L of agarose/medium solution can be required.
7. Repeat steps 1.5 – 1.6 for each bronchus in the case of several bronchi within a single specimen.
8. Put the lung on ice for polymerization of the agarose to gel for 20 – 40 min, depending on the amount of instilled agarose. Carefully touch the pleura on several sides to check whether it is hard and cool, indicating that polymerization is complete, otherwise continue to wait. Let the pathologists will cut the lung into slices, take their samples and store the lung material on ice for transportation.
9. Cut the human lung tissue into 3 – 5 cm slabs with a sharp knife.
   NOTE: Use a new blade for every lung to ensure sharpness.
10. Fill the tissue slicer with 400 mL ice-cold Earle's balanced salt solution (EBSS). Immediately cut cylindrical tissue cores out of the lung slabs using a semi-automated screwdriver with a coring tool of the preferred diameter (e.g., 8 mm; 10 mm).
    NOTE: This diameter must be equivalent to the diameter of the tissue holder inside the machine.
11. Adjust the thickness of the lung slices to the desired thickness value.
    NOTE: A thickness of approximately 250 µm is commonly used in PCLS experiments. The manufacturer of the tissue slicer recommends their Tissue Slice Thickness Gauge for verification of the slice thickness. Alternatively, whole-mount staining combined with confocal microscopy can be used to determine the slice thickness as described by Brismar et al.²⁷
12. Transfer the tissue cores into the tissue holder of the tissue slicer. Put the weight (part of the tissue holder) on top of the tissue core. Set the arm speed and blade speed to 6 on the tissue slicer. Start slicing the tissue core into PCLS.
13. Supplement 500 mL of commercially available Dulbecco's Modified Eagle Medium (DMEM): Nutrient Mixture F-12 DMEM/F12 (1:1) with 5 mL of penicillin (10,000 units/mL) and streptomycin (10,000 µg/mL). Store the culture medium for several days under sterile conditions in a refrigerator. Pre-warm only the required volume of medium.
    NOTE: Penicillin will be inactivated if kept at 37 °C for longer periods. Use serum-free and dye-free culture medium, as serum composition varies from batch to batch and dyes, such as phenol-red, can interfere with assays.
14. Fill a Petri dish (100 x 15 mm) with 25 mL ice-cold culture medium. Medium should be drained out of the tissue slicer into a beaker by opening the clamp of the glass cylinder. Transfer the slices from a beaker into the Petri dish with culture medium by using an applicator (e.g., inoculation loop). Put the Petri dish into an incubator (37 °C, 5% CO₂, 100% humidity). Allow the medium to warm up prior to washing steps.
    NOTE: All further steps are performed under sterile conditions.
15. Place a cell strainer into the Petri dish for washing the PCLS. Completely remove the culture medium with a 10-mL serological pipette through the cell strainer and add 25 mL of 37 °C pre-warmed fresh medium. Repeat this step 3 – 4 times every 30 min.
    NOTE: The cell strainer prevents the slices from being sucked into the pipette, to avoid any damage to the slices.
16. Transfer the lung slices carefully into a 24-well culture plate with a minimum of 500 µL culture medium for two slices per well. Expose the lung tissue to substances (see steps 3.1 – 3.4), e.g., for 24 h at 37 °C, 5% CO₂.
    NOTE: For the transfer, preferably use an inoculation loop and let the slices float onto the loop in order to prevent tissue damage. No further separation of the slices is needed, as only shortage of sufficient fresh medium will induce cell death in tissue slices. Slices can overlap or touch each other in the well: this has no influence on tissue viability.
17. Put all residual human material into a plastic flask with a fixative (e.g., 10% buffered formaldehyde) for at least 24 h and burn this in the disposal process.
    Caution: Formaldehyde is toxic, perform this step under a hood.

2. Histological Analysis of PCLS

1. Prepare biopsy cassettes with two foam pads soaked in fixative (e.g., 4% buffered formaldehyde). Place the PCLS between the foam pads in the biopsy cassette and immediately transfer the tissue into fixative solution. Fix the tissue overnight in 4% buffered formaldehyde.
2. Process the tissue for paraffin embedding by dehydrating samples in ethanol (70%, 90%, 100%, 100%, 100%, 100%) for 1 h each, followed by washing in xylene (3 x 1 h) and liquid paraffin (3 x 1 h) as per standard histopathology protocols.
3. Transfer the PCLS into an embedding mold. Use an inoculation loop. Embed the PCLS tissue in liquid paraffin. Make sure to place the tissue evenly in the mold when casting the paraffin block.
4. Cut the tissue block containing the PCLS with a rotary microtome until it has a flat and even surface. Take serial sections of the desired thickness (e.g., 4 µm), placing them individually on consecutively numbered coated glass slides (usually 25 – 30 sections can be taken) until the whole PCLS has been processed.
5. Stain single glass slides (every 8 – 10 sections) with hematoxylin and eosin stain (H&E) for orientation. Select sections for the experiment based on the orientation slides (the first and last 8 sections are usually incomplete).
   NOTE: (Optional) For prolonged storage, slides can be dipped in liquid paraffin for preservation.

3. Preparation of Solutions for Substances

NOTE: Prepare working solutions and controls immediately before use.

Caution: Handle substances according to safety instructions or, if unknown, as potentially harmful and follow routine safety precautions.

1. Dissolve water-soluble substances directly in culture medium. For insoluble or poorly soluble chemicals, first dissolve the substance in the appropriate solvents depending on substance solubility. Substances with limited water solubility (<0.1 mg/mL) can be dissolved, for example, in DMSO or ethanol. Non-toxic solvent concentrations should be determined by titration beforehand. Make sure the substances do not precipitate out of solution when diluted into medium.
   NOTE: HClPt and sodium laureth sulfate (SLS) solutions are prepared.
2. Prepare substance stock solutions at 100-fold of the desired highest concentration in the culture medium or solvent. Weigh 12.5 mg HClPt and 34.4 mg SLS and prepare stock solutions by dissolving the chemicals in 1 mL culture medium.
   NOTE: No solvent is required for these chemicals.
3. Prepare a final dilution of 1:100 in pre-warmed medium. In case of prior use of solvent, this approach results in the same final solvent concentration for all substance concentrations.
4. Use the final solvent concentration (e.g., 1% as described in step 3.3) for reference treatment of PCLS. No solvent control was required for the chemicals mentioned in the results section.

4. Positive and Negative References for Cytotoxicity Assays

1. For all viability assays, prepare the following positive and negative controls:
   1. Tissue control: incubate PCLS in culture medium only as a reference for untreated PCLS for 24 h at cell culture conditions (37 °C, 5% CO₂, 100% humidity).
   2. Vehicle control (if necessary): incubate PCLS with the final solvent concentration as a reference for PCLS treated with vehicle only (see step 3.4) for 24 h at cell culture conditions (37 °C, 5% CO₂, 100% humidity).
   3. Positive control: incubate PCLS with 1% detergent in buffer solution for 1 h at 4 °C.
      NOTE: If PCLS become colorless, the tissue is dead. Total L-lactate dehydrogenase (LDH) is determined in the supernatant, with an absorption of approximately 1.9 – 2.3 (see steps 6.1 – 6.4). The tissue is used for the WST-1 assay (see steps 5.1 – 5.5), with an absorption of approximately 0.

5. Measurement of Chemically-induced Cytotoxicity in Human PCLS by WST-1 Assay

NOTE: The WST-1 assay is performed in a 24-well plate with two PCLS per well. Preferably, use duplicates for each parameter and pool the results of these duplicates after measurement.

1. Prepare the working solution by diluting the WST-1 reagent in medium immediately before starting. The required amount of working solution is 250 µL/well of a 24-well plate. Therefore, mix 25 µL of the reagent with 225 µL of the culture medium for one well.
2. After incubation of PCLS from step 1.16, discard or use the supernatant for cytokine measurements or other tests such as the LDH assay. The remaining tissue is used for the next step.
3. Pipet 250 µL of the working concentration of the WST-1 reagent per well and incubate the plate at 37 °C and 5% CO₂ for 1 h. Ensure that the PCLS are fully covered by the WST-1 reagent during incubation.
4. Place the plate on an orbital shaker (200 rpm) and shake carefully for 30 s to ensure thorough mixing of the WST-1 reagent. Take a new flat-bottom 96-well plate and pipet 100 µL in duplicates from the supernatant of each well of the 24-well plate.
5. Measure the absorption of each well at 450 nm (reference: 630 nm) using a microplate reader. Subtract the absorption at 630 nm from 450 nm. These values will be used for the calculation in step 5.6).
   NOTE: Reliable data for cytotoxicity assessment can be obtained only from viable tissue. Therefore, these acceptance criteria published by Hess et al.¹⁵ should be met in each experiment. If these criteria are not met, the experiment should be repeated.
6. Absorption of the untreated medium control should be above 0.6, otherwise the experiment should be repeated. If the data meet this requirement, set the absorption value of the tissue control to 100% and calculate the WST-1 reduction of treated samples in relation to the tissue control.

6. LDH Assay

Note: The LDH assay is performed in a 96-well plate with 100 µL culture supernatant in total after the post-incubation period.

1. After incubation of the PCLS with or without the test agents, take 50 µL of supernatant from step 1.16 and transfer it in duplicates into a new 96-well plate. This generates duplicates from each treated well of a 24-well plate.
2. Prepare the working solution of LDH reagent immediately before the assay. The working solution consists of the catalyst solution (lyophilisate dissolved in 1 mL bidistilled water) and dye solution supplied by the manufacturer. For one 96-well plate, thoroughly mix 125 µL of catalyst solution with 6.25 mL of dye solution.
   Caution: Do not expose the solution to direct light.
3. Pipet 50 µL of the working solution into each well already containing 50 µL of supernatant and incubate the plate for 20 min at RT in the dark. No further mixing is required.
4. Measure the absorption of each well at 492 nm (reference: 630 nm) using a microplate reader. Subtract the absorption at 630 nm from 492 nm. These values will be used for the calculation in step 6.5.
   NOTE: Reliable data for cytotoxicity assessment can be obtained only from viable tissue. Absorption of the detergent-treated control should be over 1.
5. For analysis, set the absorption value of the positive control from step 4.1 as 100% and calculate the LDH release in treated samples in relation to the positive control (i.e., maximum LDH release).

7. Microscopic Viability Assay with Confocal Laser Scanning Microscope (cLSM)

Note: For the microscopic viability assay, two of the normal 250-µm-thick PCLS are stained in 250 µL/well of a 24-well plate. As each slice is imaged separately, no further duplicates are required.

1. After incubation of PCLS with or without test items, discard the supernatant or use it for cytokine measurements or other tests such as the LDH assay. Use the remaining tissue for the procedure described here.
2. Prepare the working dilution of calcein-AM and ethidium homodimer 1 (EthD-1) with a working concentration of both reagents of 4 µM in culture medium. For this purpose, add 1 µL of calcein-AM (4 mM) and 2 µL of EthD-1 (2 mM) to 997 µL culture medium. This volume is required to stain 4 wells with two PCLS each.
   NOTE: Avoid exposure of the reagents to direct light.
3. Pipet 250 µL of the working dilution in each well and incubate the 24-well plate for 45 min at RT in the dark. Place the plate on an orbital shaker at 150 rpm during the incubation to ensure better exposure of the tissue to the staining solution.
4. After 45 min, discard the staining solution and wash the PCLS with 1 mL buffer solution through shaking at 150 rpm for 3 – 5 min at RT in the dark. Repeat this step twice.
5. Apply 500 µL of buffer solution to each well containing the stained PCLS to avoid autofluorescence from culture medium. For microscopic assessment, perform at least two randomly distributed z-stacks with a cLSM.
6. Click on the Ocular tab to choose a 10X/0.3 objective and click on Online to use the cLSM as a standard light microscope to find the surface of PCLS. Click on Offline to exit the ocular setting.
7. Click on the Acquisition tab and turn on the appropriate lasers for the fluorophores.
   NOTE: We used an argon laser with a wavelength of 488 nm for the polyanionic dye calcein (excitation wavelength of 495 nm) and a HeNe laser with a wavelength of 543 nm for detection of the EthD-1/DNA complex (excitation wavelength of 533 nm). The argon laser should generally run at 30 – 50% of the maximum current to enable a tube current of around 5.7 A, as this prolongs laser lifetime significantly.
8. Click on Light Path under the Acquisition tab and set up the necessary filters and mirrors for the experiment.
   NOTE: In the present study, a filter-based system was used with a main dichroic beam splitter (e.g., HFT 488/543) separating the excitation and emission light. Through a reflecting mirror this light is directed to the secondary dichroic beam splitter (e.g., NFT 545) to further separate the light emitted from the sample into two channels.
9. Set up band pass filters (e.g., BP 505-530 for the calcein signal and BP 560-615 for the EthD-1/DNA complex signal) that transmit only a certain range of wavelengths and assign the calcein signal to channel 2 and the EthD-1/DNA complex signal to channel 3.
10. Check the Z-Stacks box to set the upper and lower limits for the microscopic volume. Press the Live button to see a live view of the corresponding layer on the screen. Move the focus up or down to find the surface of the PCLS with a sharp signal.
11. Click on the Channels subsection and check the box Show all. Lock up the table by pressing on the button of the corresponding channel underneath the live image and activate the channel color.
    NOTE: A blue color in the live image will indicate that there is no signal, whereas a high signal will appear in white and an overexposed signal will appear in red.
12. Set the pinhole for the red EthD-1 channel to 1 Airy unit for the best trade-off between efficiency of light collection and optical sectioning and adjust the calcein channel accordingly. Increase the detected signal of the gain such that it appears white but not red in the live image with activated channel color lockup table.
    NOTE: The master gain should not exceed 600 units.
13. Increase or decrease the digital offset to adjust the background such that it appears in blue in the live image with activated channel color lockup table.
14. Click on the Z-Stack tab under Multidimensional Acquisition and use the focus to shift to a z-layer of interest. Press Set First to save this position for later acquisition. Slowly shift the focus up or down until a range of 30 µm has been reached and press Set Last to save.
15. Click on Live once again to deactivate the live image and press on Start Experiment to start the imaging.
    NOTE: Viable tissue is detected through fluorescence of the polyanionic dye calcein. Dead cells are discriminated by complex formation of EthD-1 and nucleic acids.

8. Image Rendering

Note: The computer recommendation of the image rendering software must be taken into account, as this program requires the appropriate hardware.

1. Load the LSM file acquired from microscopic viability staining of PCLS (see Section 7) into the image rendering software. Save it as .ims file before proceeding. Set all parameters on the tissue control and apply these to all images. No further changes are permitted.
2. Use the Volume button for surface reconstruction of viable tissue (calcein staining) (Supplementary Figure 1A) and the Spots button for dead cell nuclei (Supplementary Figure 1H). When rendering one channel, switch off the other channel in the display adjustment window.
3. Start by rendering the volume of vital tissue: set the channel of the surface, process the entire image, set the smooth factor to 0.5 µm, and use absolute intensity for threshold (Supplementary Figure 1B, C). Press the blue forward arrow.
4. Adjust the absolute intensity threshold of the reconstructed volume; noise and background intensity should be subtracted. Surface overlay is shown in grey and the accuracy of surface coverage should be checked (Supplementary Figure 1D). After completing the adjustment, press the blue forward arrow. Most of the time this step is required for calculation of the settings. If the software is not able to calculate the volume, increase the smooth factor.
5. In the last step, choose a filter. Use the sphericity filter (with approximately 10 µm) for surface rendering to filter out macrophages in the alveolar space (Supplementary Figure 1E). Press the green forward button.
6. Optically adjust the output image and export statistics as .xls files. The total volume (sum) will be used for further analysis (Supplementary Figure 1F, G). Save the settings and use them for all further analyses of z-stacks taken on the same day with the same cLSM settings (Supplementary Figure 1I).
7. Reconstruct the red channel (spots) for dead cell nuclei. Randomly measure the dot size in µm scaling in the surpass window; dot size is approximately 5 µm. Mark Enable and set the dot size. Check whether all dots are marked and each dot is counted only once. Correct manually if required (Supplementary Figure 1B, H, J). Press the blue forward arrow.
8. Press the green forward button to let the program calculate/count the total number of spots according to the parameters set and export the result as .xls file (Supplementary Figure 1K, L, M). For graphical or statistical analysis of the image set the number of calculated dead cell nuclei in ratio to the volume of vital tissue.

9. Measurement of Cytokines and Chemokines in Human PCLS

Note: The cytokine and chemokine immune response can be measured extracellularly in the culture supernatant and intracellularly in tissue lysates after the incubation time. ELISA is measured in duplicates in a 96-well plate with 100 µL/well.

1. Prepare a 1% detergent solution by mixing 5 mL of detergent with 495 mL of buffer solution. The solution can be stored at RT for several months.
2. Mix the 1% detergent solution with protease inhibitor (PI) at a ratio of 1:500. The required amount depends on the culture plate; for example, use 500 µL/well for a 24-well plate. For one well, mix 1 µL of PI with 499 µL of detergent solution.
3. Prepare tubes with 1 µL of PI solution and collect 500 µL of culture supernatant in these tubes. Immediately replace the medium in the 24-well plate by 500 µL detergent solution containing PI as prepared in step 9.2. Lyse tissue for 1 h by placing the 24-well plate in at 4 °C. Pipet the tissue lysate in a new tube and store these tubes at -80 °C until further analysis.
   NOTE: The ELISA protocol highly depends on the manufacturer and the manufacturer's instructions should be followed. A standard ELISA protocol is described in the following.
4. Perform the ELISA to measure cytokine levels in supernatant or lysate according to the manufacturer's instructions. To measure IL-1α and TNF-α, coat a96-well plate by pipetting 100 µL of the capture antibody (for dilution follow the manufacturer's instructions) and incubate overnight at RT.
5. Prepare washing buffer by dissolving a washing buffer tablet in 1 L bidistilled water. Remove the capture antibody and pipet 400 µL washing buffer into each well. Replace this volume three times. Block the plate by adding the blocking solution (e.g., 1% BSA in buffer solution, according to the manufacturer's instructions). Incubate at RT for 1 h.
   NOTE: Standard commercially available ELISAs require 2 x 100 µL of tissue supernatant or lysate. Samples should be thawed once and just prior to use. Depending on the sensitivity of the assay and on the released cytokine, samples must be diluted prior to measurements. Therefore, dilute the sample in the reagent provided by the assay.
6. Remove the blocking solution and add diluted samples for the standard curve (for dilution follow the manufacturer's instructions) and 100 µL of tissue supernatant or 100 µL of tissue lysate in duplicates collected in step 9.3. Incubate for 2 h at RT.
7. Remove the samples and pipet 400 µL washing buffer into each well. Replace this volume three times. Pipet 100 µL biotinylated detection antibody (for dilution follow the manufacturer's instructions) and incubate for 2 h at RT.
8. Remove the antibody and pipet 400 µL washing buffer into each well. Replace this volume three times. Add 100 µL/well of HRP-labeled streptavidin (for dilution follow the manufacturer's instructions) and incubate for 20 min at RT (check the manufacturer's instructions).
9. Remove streptavidin and pipet 400 µL washing buffer into each well. Replace this volume three times.
10. Add 100 µL of the substrate (recommended by the manufacturer) and incubate for 20 min in the dark (check the manufacturer's instructions).
    NOTE: This step is light-sensitive. Avoid light exposure of the substrate and plate.
11. Stop the reaction by adding 50 µL stop solution (1 M H₂SO₄). Measure the absorption of each well at 450 nm (reference: 570 nm) using a microplate reader. Subtract the absorption at 570 nm from 450 nm.
    NOTE: Untreated and/or vehicle tissue controls serve as negative controls. To induce an immune response in lung tissue, use appropriate stimulation (e.g., 100 ng/mL lipopolysaccharide (LPS)) as a positive control. Incubate two wells with two PLCS per well with 100 ng/mL LPS in culture medium, for example, for 24 h, and collect the supernatant and tissue lysate as described in step 9.3.
    NOTE: The measured results are accepted, if the absorption of the highest standard is equal to or above 1.0; otherwise the measurement needs to be repeated. The standard curve should follow the sigmoidal dose-response curve fitting.
12. Cytokine concentrations (pg) determined by ELISA in step 9.11 are normalized to the total protein content determined in tissue lysate of each sample (see Section 10).

10. Measurement of Total Protein Content in Human PCLS

Note: The PCLS need to be lysed to measure total protein concentration. The tissue lysates from step 9.3 are used for this step. The assay is performed in a flat 96-well plate with 25 µL/well of tissue lysate, pipetted in duplicates.

1. Prepare a 7-point BSA standard with BSA concentrations of 2,000 µg/mL, 1,500 µg/mL, 1,000 µg/mL, 750 µg/mL, 500 µg/mL, 250 µg/mL, and 125 µg/mL. Apply 25 µL in duplicates for each standard and use 25 µL buffer solution as a blank on a 96-well plate (use a plate with a plate cover).
2. Pipet 25 µL of the lysates (see step 9.3) from each well in duplicates into a 96-well plate. In total, 50 µL of each well is required. Prepare the working reagent of BCA solution by diluting BCA reagent B 1:50 in BCA reagent A. For a 96-well plate use 400 µL of reagent B and 20,000 µL of reagent A.
3. Carefully pipet 200 µL of the working reagent into each well, avoiding air bubbles. Place the plate on an orbital shaker (150 rpm) for 30 s to ensure proper mixing of lysates and working reagent, and put a plate cover on the plate. Incubate the plate at 37 °C for 30 min in the dark. Measure the absorption of each well at 540 – 590 nm using a microplate reader.
   NOTE: The measurement results are accepted, if the absorption of the highest BSA standard is equal to or above 0.8; otherwise the measurement needs to be repeated. The standard curve should follow the sigmoidal dose-response curve fitting.
4. For analysis, calculate the standard curve using a 4-parameter Marquardt equation after subtraction of the blank. Calculate the protein concentrations of unknown samples by using the standard curve fit.