Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.
Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated β-galactosidase (SA-β-gal), the DNA synthesis marker 5-ethynyl-2′-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (γH2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.
A variety of stimuli can trigger cellular senescence, causing cells to enter a state of stable cell cycle arrest. These stimuli include intrinsic signaling changes or extrinsic stresses. Intrinsic signals include progressive telomere shortening, changes in telomere structure, epigenetic modification, proteostasis disorders, mitochondrial dysfunction, and activation of oncogenes. Extrinsic stresses include inflammatory and/or tissue damage signals, radiation or chemical treatment, and nutritional deprivation1,2,3,4. Among distinct types of senescence, the most commonly seen and well-studied are replicative senescence, oncogene-induced senescence (OIS), radiation-induced senescence, and therapy-induced senescence (TIS). OIS is an acute cellular response to genotoxic damage caused by replicative stress generated by aberrant oncogene activation and can to some extent prevent the pathological progression from a preneoplastic lesion to a full-blown tumor. TIS happens when tumor cells are stressed by chemotherapeutic drugs or ionizing radiation5,6.
Senescence is considered a double-edged sword in pathology due to its highly dynamic nature. It was initially described as a beneficial tumor-suppressive mechanism to remove damaged cells from the circulating pool of dividing cells, safeguarding the normal function of organs and inhibiting tumor growth7,8,9. However, emerging evidence has suggested a dark side of senescence. Senescent cells secrete proinflammatory cytokines, known as senescence-associated secretory phenotype (SASP), leading to fibrosis and malfunctional organs and promoting tumor initiation and progression10. Moreover, senescent cancer cells undergo epigenetic and gene-expression reprogramming in parallel with chromatin remodeling and activation of a sustained DNA-damage response (DDR)11,12, newly acquiring new cancer-stem-cell properties3. Although senescence-capable tumors respond better to therapeutic intervention compared to senescence-incapable ones13, the persistence of senescent cells may lead to a poor long-term prognosis if they are not effectively identified and eliminated by senolytic drugs5. Either way, a reliable method to assess senescence is of significant clinical interest, not only for the prognosis of therapy treatment but also for the development of novel strategies targeting senescent cells.
Regardless of different triggers, senescent cells exhibit some common features, including enlarged, flattened, multinucleated morphology with big vacuoles, significantly expanded nuclei, formation of H3K9me3-rich senescence-associated heterochromatin (SAHF) in the nucleus, persistent accumulation of DNA damage marker γH2AX foci, activated p53-p21CIP1 and Rb-p16INK4a cell cycle regulatory mechanisms, stable G1 cell cycle arrest, massive induction of SASP, and elevated senescence-associated β-galactosidase (SA-β-gal) activity14. Since no single marker is sufficient to define senescence, enzymatic staining for SA-β-gal activity, which is considered the gold standard for senescence detection, is usually combined with immunohistochemical staining for H3K9me3 and Ki67 to detect TIS15. However, chemical chromogenic-based SA-β-gal is difficult to quantify. Here, we combined 5-dodecanoylaminofluorescein-di-β-D-galactopyranoside (C12FDG) fluorescence-based SA-β-gal (fSA-β-gal) detection with immunofluorescent staining for γH2AX and EdU-incorporated DNA to identify C12FDG+EdU-γH2AX+ senescent cells using the advanced imaging flow cytometer system, which combines the speed, sensitivity and detailed single-cell images with spatial information that cannot be provided by flow cytometry and microscopy. This method enables rapid generation of high-resolution images allowing for the positioning and quantification of fluorescent signals within cells, while licensing the swift analysis of multiple samples by building standard pipelines.
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1. DLBCL cell lines with mafosfamide or daunorubicin treatment to induce cellular senescence
NOTE: The protocol also works for adherent cancer cells. Depending on cell size, seed 1-2 × 105 cells into one well of a 6-well plate and incubate the plate in a 5% CO2, 37 °C incubator overnight before treatment. The protocol steps are the same as for suspension cells but with two exceptions. First, cells need to be trypsinized off the plate after step 3.4. Second, wash steps are performed without centrifugation before trypsinization.
- Count DLBCL cells and seed 1 × 106 cells/mL in 4 mL of medium per well into a 6-well culture plate. Cultivate DLBCL cells in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin.
- Add MAF (5 µg/mL) or DN (20 ng/mL) into the cell culture and gently rock the plate to mix.
NOTE: MAF and DN are not stable. After dissolving in DMSO, the stock solution should be aliquoted and stored at -20 °C. Freeze/thaw cycles should be avoided.
- Incubate the plate in a 5% CO2, 37 °C incubator for 3 days.
- After a 3 day incubation, collect the DLBCL cells in 15 mL sterile centrifuge tubes and spin at 100 × g, 4 °C for 5 min.
- Discard the supernatant, resuspend the cell pellets with 4 mL of fresh medium, and add the suspensions back into the 6-well plate.
- Cultivate the cell plate in a 5% CO2, 37 °C incubator for another 2 days before harvesting for analysis.
2. Prepare solutions for staining (Table 1)
3. Stain DLBCL cells with different senescence markers
NOTE: Cell samples stained with individual markers (i.e., pacific blue-EdU, C12FDG, or Alexa Fluor 647-γH2AX) are prepared to generate a compensation matrix to correct the fluorescence spillover during measurement. Although highly suggested, this step could be suspended when there is an omittable overlap (compensation coefficient value ≤ 0.1) of the emission spectra among different fluorophores. However, users must determine standardized compensation steps when using different instruments and fluorescent panels.
- Add 10 mM EdU solution at a ratio of 1:1,000 into the DLBCL cell culture generated from step 1.6 (final EdU concentration is 10 µM). Gently rock the plate to mix and incubate in a 5% CO2, 37 °C incubator for 3 h.
- Take the plate out of the incubator and add 100 mM chloroquine solution to a final concentration of 75 µM (see discussion). Gently rock the plate to mix and incubate in a 5% CO2, 37 °C incubator for 30 min.
- Take the plate out of the incubator and add 20 mM C12FDG solution at 1: 1,000 to a final C12FDG concentration of 20 µM. Gently rock the plate to mix and incubate in a 5% CO2, 37 °C incubator for 1 h.
- Take the plate out of the incubator and add 100 mM 2-phenylethyl-β-D-thiogalactoside (PETG) solution at 1:50 to stop the fSA-β-gal staining (final PETG concentration of 2 mM). Gently rotate the plate to mix.
- Transfer the cells to 15 mL sterile centrifuge tubes and spin at 100 × g, 4 °C for 5 min. Discard the supernatant and wash the cells with 4 mL of phosphate-buffered saline (PBS).
- Repeat the PBS washing step. Discard the supernatant and resuspend the cell pellets in 500 µL of 4% paraformaldehyde fixation solution.
- After 10 min incubation at room temperature, centrifuge at 250 × g, room temperature for 5 min. Discard the supernatant and wash cells with 4 mL of PBS.
- Repeat the PBS washing step. Discard the supernatant, resuspend the cell pellet in 200 µL of saponin permeabilization buffer and transfer the suspension to a new 1.5 mL tube. After 10 min incubation at room temperature, centrifuge at 250 × g, room temperature for 5 min.
- Discard the supernatant and resuspend the cell pellets in 200 µL of primary antibody solution (1:500 γH2AX antibody in antibody incubation solution). Incubate at 4 °C overnight in the dark.
- Centrifuge the tubes at 250 × g, 4 °C for 5 min. Discard the supernatant and wash with 100 µL of saponin wash solution.
- Repeat the washing step an additional two times. Discard the supernatant and resuspend the cell pellets in 500 µL of EdU detection cocktail.
- Incubate at room temperature for 30 min in the dark. Centrifuge at 250 × g, room temperature for 5 min. Discard the supernatant and wash with 1 mL of saponin wash solution.
- Repeat the washing step two times. Discard the supernatant and resuspend the cell pellets in 20-50 µL of PBS. Proceed to section 4 for sample measurement.
4. Imaging senescence markers using the imaging flow cytometer system
- Empty the waste fluid bottle. Check the levels of speed beads, sterilizer, cleaner, debubbler, sheath, and rinse reagents (deionized water) to ensure sufficient fluids before turning on the instrument (see the Table of Materials).
- Turn on the instrument and imaging software (see the software interface in Figure 1A).
- Click on the Startup button to initialize fluidics and system calibration.
NOTE: This procedure takes approximately 45 min.
- Set the magnification to 40x, set fluidics speed to low, and turn on the lasers needed in the experiment. Turn on 405 nm, 488 nm, and 642 nm lasers to measure EdU-pacific blue, C12FDG, and Alexa Fluor 647-γH2AX (or Alexa Fluor 647-Ki67), respectively. Set Ch6 for scatter channel, and Ch1 and Ch9 for brightfield.
- Start with the sample expected to have the highest fluorescence to set up the intensity of the lasers. Gently flip the sample tube to mix. Open the tube lid, and insert the sample tube onto the dock. Click on Load to start.
- Open a scatter plot and select the features Area_M01 and Aspect Ratio_M01 for the X- and Y-axes, respectively. Set a gate above aspect ratio of 0.5 to exclude doublets and cell aggregates below and on the right side, and the speed bead population on the left side (Figure 1B).
- Open a histogram plot and select the feature Gradient RMS_M01_Ch01 for the X-axis. Choose the singlet population and set the gate to select for focused cells (Figure 1C).
NOTE: The imaging system will automatically use speed beads to adjust the focus for imaging. However, it is recommended to select the right half of the histogram peak for the best-focused population.
- Open a histogram plot and select Raw Max Pixel Intensities for X-axis for each color channel (Ch2, 7, and 11). Adjust the laser powers (i.e., 488 nm, 405 nm, and 642 nm lasers for Ch2, 7, and 11, respectively), so each fluorochrome has a Raw Max Pixel value between 100 and 4,000 to avoid oversaturation.
NOTE: For this experiment, the laser power setting is 50 mW, 200 mW, and 50 mW for lasers 488 nm, 405 nm, and 642 nm, respectively.
- Choose the focused population to record and click on Acquire to measure the DLBCL samples with consistent settings. When changing samples for measurement, click on Return to recover the sample tube. Press Load to discard the sample.
- After all the samples are measured, turn off the brightfield and scatter laser. Measure single-color control samples to generate a compensation matrix.
- Click on Shutdown to close the imaging system.
- Analyze the data in the image analysis software.
- Use the Spot Wizard tool of the image analysis software to automatically count and quantify nuclear γH2AX foci in live-cell images. Select two cell populations (one with high and one with low spot count) to train the spot wizard for further automatic spot count analysis.
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A compensation matrix was generated using image analysis software by loading recorded data of single-color control samples. As shown in Supplemental Figure S1, a non-negligible (coefficient value ≥ 0.1) light spillover from EdU to C12FDG was detected with crosstalk coefficient value 0.248, while the crosstalk among other channels was not significant. Four different DLBCL cell lines were treated with 5 µg/mL MAF or 20 ng/mL DN to induce cellular senescence and analyzed using either conventional SA-β-gal staining or the imaging flow-cytometry method.
More than 70% of KARPAS422, WSU-DLCL2, and OCI-LY1 cells entered senescence status, indicated by positive SA-β-gal, while SU-DHL6 was fully resistant to senescence induction by MAF and DN (Supplemental Figure S2A). Notably, MAF and DN also induced cell death in all DLBCL cells, although not dramatically (Supplemental Figure S2B). Using the imaging flow-cytometry method, single-cell images, and flow cytometry-based quantification showed an increased C12FDG+EdU-γH2AX+ senescent population in KARPAS422, WSU-DLCL2, and OCI-LY1 but not in SU-DHL6 cells (Figure 2A-D), which was consistent with the conventional SA-β-gal staining results. However, the percentage of the C12FDG+EdU-γH2AX+ senescent population was significantly lower than the conventional SA-β-gal staining method (Figure 2 and Supplemental Figure S2).
Moreover, imaging flow-cytometry analysis showed diverse senescence inducibility among different DLBCL cell lines, which was not clearly distinguished by conventional SA-β-gal staining. The percentage of C12FDG+EdU-γH2AX+ senescent OCI-LY1 cells (i.e., 43.2% and 31.9% with MAF and DN, respectively), was significantly lower than that of KARPAS422 (62.2% and 73.8% with MAF and DN, respectively) and WSU-DLCL2 (60% and 58.1% with MAF and DN, respectively) (Figure 2). Importantly, imaging-based analysis presented significantly higher numbers of γH2AX foci in KARPAS422, WSU-DLCL2, and OCI-LY1 but not in SU-DHL6 cells (Figure 3A,B).
Figure 1: Operating software interface and instrument setup. (A) Imaging flow-cytometer software interface. The live-cell image panel (top left) contains live-cell images of all 12 image channels and image adjustment tools (e.g., zoom, cell population selection, color change, mask, and image properties adjustment). The flow cytometry analysis area (bottom left) contains a toolbar for flow cytometry analysis, such as histogram plot, scatter plot, various region gating, layout, and population statistics. The Control panel (right) contains menus for data acquisition and saving, illumination setting, magnification, fluidics speed control, focus, and centering. (B) Selection of single-cell population. (C) Selection of focused cell population. Please click here to view a larger version of this figure.
Figure 2: Visualization and quantification of senescent cells on a single-cell level. Representative images (left panel) and flow cytometry analysis of C12FDG+EdU-γH2AX+ senescent cells (right panel) of (A) KARPAS422, (B) WSU-DLCL2, (C) OCI-LY1, and (D) SU-DHL6 cells treated with 5 µg/mL MAF or 20 ng/mL DN for 5 days and stained for fSA-β-gal, EdU, and γH2AX. DMSO-treated cells were used as control. Abbreviations: C12FDG = 5-dodecanoylaminofluorescein-di-β-Dgalactopyranoside; EdU = 5-ethynyl-2′-deoxyuridine; γH2AX = phosphorylated form of histone H2AX; DMSO = dimethyl sulfoxide; MAF = mafosfamide; DN = daunorubicin; fSA-β-gal = fluorescence-based senescence-associated β-galactosidase. Please click here to view a larger version of this figure.
Figure 3: Imaging-based quantification of nuclear γH2AX foci in senescent cells. (A) Representative images of γH2AX foci in DLBCL cells (see text of Figure 2 for treatment and staining details). (B) Frequency (left) and quantification (right) of γH2AX foci counts in DLBCL cells. Abbreviations: γH2AX = phosphorylated form of histone H2AX; DMSO = dimethyl sulfoxide; MAF = mafosfamide; DN = daunorubicin. Please click here to view a larger version of this figure.
|10 mM EdU solution in DMSO||Store at -20 °C.|
|20 mM C12FDG solution in DMSO||Store at -20 °C.|
|100 mM chloroquine solution in deionized water||Store at -20 °C.|
|100 mM PETG solution in deionized water||Store at -20 °C.|
|Fixation solution: 4% PFA (paraformaldehyde) in PBS||freshly prepared; CAUTION: PFA is harmful. Use with appropriate precautions.|
|Permeabilization buffer: 1% saponin (w/v), 3% BSA (w/v) in PBS, freshly prepared.||freshly prepared|
|Antibody incubation solution: 0.1% saponin (w/v), 3% BSA (w/v) in PBS||freshly prepared|
|Wash solution: 0.1% saponin (w/v), 0.5% BSA (w/v) in PBS||freshly prepared|
|EdU detection cocktail: Dilute CuSO4 (100 mM) at 1:50, pacific blue azide solution at 1:200 and reaction buffer additive (200 mg/mL) at 1:100 in PBS||freshly prepared|
Table 1: Solutions for staining.
Supplemental Figure S1: Compensation matrix of EdU-pacific blue, C12FDG-Fluorescein, and AF647-γH2AX. Abbreviations: C12FDG = 5-dodecanoylaminofluorescein-di-β-Dgalactopyranoside; EdU = 5-ethynyl-2′-deoxyuridine; γH2AX = phosphorylated form of histone H2AX; AF647 = Alexa Fluor 647. Please click here to download this File.
Supplemental Figure S2: Therapy-induced senescence of DLBCL cell lines. (A) Conventional SA-β-gal staining (left panel) and quantification (right panel) of indicated DLBCL cell lines treated with 5 µg/mL MAF or 20 ng/mL DN. Scale bars = 50 µm. (B) Percentage of cell death analyzed by flow cytometry (left panel) and quantification (right panel) of DLBCL cell lines as in A. Cells were stained with ghost dye-pacific blue at room temperature for 15 min. Abbreviations: DMSO = dimethyl sulfoxide; MAF = mafosfamide; DN = daunorubicin; SA-β-gal = senescence-associated β-galactosidase. Please click here to download this File.
Supplemental Figure S3: Representative images and flow cytometry analysis. (A) Representative images and flow cytometry analysis of C12FDG+EdU-Ki67+ senescent cells (B) of KARPAS422 and WSU-DLCL2 cells treated with MAF for 5 days and stained for fSA-β-gal, EdU, and Ki67. DMSO-treated cells were used as control. Abbreviations: C12FDG = 5-dodecanoylaminofluorescein-di-β-Dgalactopyranoside; EdU = 5-ethynyl-2′-deoxyuridine; DMSO = dimethyl sulfoxide; MAF = mafosfamide; DN = daunorubicin; fSA-β-gal = fluorescence-based senescence-associated β-galactosidase. Please click here to download this File.
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This method examined the senescence-entering capability of four different DLBCL cell lines upon chemotherapy treatment, with bright-field imaging and flow cytometry-based quantification. On a single-cell level, we successfully detected major C12FDG+EdU-Ki67+ senescent populations in treated KARPAS422 and WSU-DLCL2 cells, and to a lesser extent in OCI-LY1 cells, while the SU-DHL6 cell line was resistant to the treatment. The difference in senescence-entering capability among cell lines may be accounted for by their distinct genomic defects16,17. However, inappropriate cell culture density or drug concentration may also influence senescence inducibility. Serial dilutions of cell density and drug concentration should be carefully selected for a more comprehensive examination. Notably, this method was sensitive enough to accurately detect the naturally occurring senescent population in DLBCL cells without additional genotoxic stimulation (C12FDG+EdU-Ki67+ DMSO-treated group in Figure 3).
The level of β-galactosidase activity and the rhythm of DNA synthesis vary significantly among different cell types. The stated incubation time (i.e., 3 h for EdU and 1 h for C12FDG) is generally sufficient to discriminate positive and negative staining in vitro. However, in special circumstances (e.g., slow-growing cells or cells with extremely high β-galactosidase activity), it might be necessary to prolong EdU labeling or shorten C12FDG staining for better discrimination, respectively. It is suggested to perform a pilot experiment with a serial incubation time to optimize experimental conditions (e.g., 30 min increase or 15 min decrease for EdU or C12FDG, respectively). To better distinguish the differences in fSA-β-gal between proliferating cells and their senescent counterparts, preincubation with chloroquine or bafilomycin A1 is recommended to lower the basal lysosomal activity by inducing lysosomal alkalinization18,19. For cells with relatively low lysosomal activity, the preincubation step can be omitted.
Intact cell membrane structure is essential for fSA-β-gal detection. In this method, we used EdU instead of BrdU (5-bromo-2'-deoxyuridine) to measure DNA synthesis. Compared to BrdU incorporation, EdU needs a relatively mild staining condition, which preserves the intact membrane structure and allows co-staining with fSA-β-gal. Notably, antibody-mediated detection of intracellular or nuclear proteins (e.g., γH2AX) described here also needs permeabilization of the cell membrane to allow antibody entry. Here, saponin was exchanged for the strong detergent Triton X-100 to avoid quenching of fSA-β-gal signal.
Chemotherapeutic treatment not only triggers senescence but also cell death in DLBCL cells. Dead cells may have a non-negligible impact when quantifying the senescence population as they may result in false positives or false negatives during staining. Although most of the cell debris and small-size dead cell bodies will be excluded during multiple washing and centrifugation steps, the remaining dead cell population may still have a non-negligible influence. It is suggested to optimize drug concentration to avoid drastic cell death. In addition, cell viability staining (e.g., ghost dye) can be employed to facilitate discriminating dead cell population and quantifying senescence population more stringently.
The advanced imaging flow cytometry system used in this method is a multi-channel imaging flow cytometer, which allows for quantifying flow-cytometry-based measurements and inspecting multi-color images of single cells in parallel. It provides a quick, efficient, and accurate quantitative solution for senescence detection, and in the meantime, generates single-cell resolution, multi-color images with spatial information of multiple markers. However, some limitations persist. For instance, the image resolution is limited compared to the high-end microscope, which impedes a comprehensive analysis of subcellular localization when tracking proteins located in smaller organelles other than the nucleus. Compared to sophisticated operating and analyzing software of a standard flow cytometer, it is less convenient and less efficient to set up or change population gating strategies. Quantification and statistical analysis are also more complicated.
fSA-β-gal staining has significant advantages over conventional SA-β-gal for rapid staining and unbiased quantification and has the potential for the effective classification of senescent cells. This technique has been successfully applied to screen for senescent cells re-entering the cell division cycle3,18,19,20. In this method, we took a step further to combine fSA-β-gal with EdU and γH2AX for identifying senescent cells more accurately and reliably. Notably, although fSA-β-gal, EdU, and γH2AX were chosen as senescence-associated markers in this method, other reliable senescence markers can be easily adapted to substitute or add to the three markers. Due to the high interest in senescence in academia, many reliable senescence-associated markers have been identified, such as elevated cytoplasmic reactive oxygen species (ROS) production, secreted SASP factors, downregulated Ki67, loss of nuclear envelop protein Lamin B1, and increased heterochromatin marker H3K9me321,22. Supplemental Figure S3 showed successful detection of C12FDG+EdU-Ki67- senescent populations in TIS KARPAS422 and WSU-DLCL2 cells.
Moreover, there are many senescence cell surface markers available to be tested using this method, such as ICAM-1, NOTCH1, NOTCH3, DDP4, CD36, and uPAR. Detection of cell surface markers only requires a short antibody incubation time without cell fixation and permeabilization, which will shorten the staining procedure into a 6 h workflow in combination with fSA-β-gal and EdU, or even into a 2 h experiment in combination with fSA-β-gal only, and more importantly, it allows live-cell imaging. The modified method could potentially serve as a swift, reliable, and feasible approach in the clinic to identify senescence cells in vivo, not only for diagnostic and prognostic evaluation but also for supporting further senolytic intervention.
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The authors have no conflicts of interest to disclose.
This work was supported by a grant to Yong Yu from Johannes Kepler University Linz (BERM16108001).
|Alexa Fluor 647 anti-H2A.X Phospho (Ser139) Antibody||Biolegend||613407|
|Anti-Ki-67 Mouse Monoclonal Antibody (Alexa Fluor 647)||Biolegend||350509|
|C12FDG (5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside)||Fisher Scientific||11590276|
|Chloroquin -diphosphat||Sigma aldrich||C6628|
|Cleanser (Coulter Clenz)||Beckman Coulter||8546929|
|Click-iT EdU Pacific Blue Flow Cytometry Assay Kit||Thermo Scientific||C10418|
|Debubbler (70% Isopropanol)||Millipore||1.3704|
|Image Analysis software (Amnis IDEAS 6.3)||Luminex||CN-SW69-12|
|Instrument and imaging software (Amnis ImageStreamX Mk II Imaging Flow Cytometer System and INSPIRE software)||Luminex||100220|
|PETG (2-Phenylethyl-β-D-thiogalactosid)||Sigma aldrich||P4902|
|SpeedBead Kit for ImageStream||Luminex||400041|
|Sterilizer (0.4-0.7% Hypochlorite)||VWR||JT9416-1|
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