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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Streszczenie

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.

This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Wprowadzenie

The use of animals for experiments has limitations. Animal studies cannot accurately mimic disease progression in humans, and animals and humans do not have identical responses to pathogens. Additionally, restrictions in animal experimentation due to concerns for animal suffering and ethical problems1,2 increasingly constrain research programs. In vitro systems have been widely developed to circumvent the use of animals; moreover, the use of human cells has made in vitro models more relevant for the pathophysiological and therapeutic investigation. Conventional monolayer (2D) cell cultures are widely used because they mimic human tissues to some degree. However, 2D monocultures fail to mimic human organs, and 2D monocultures are unable to simulate the complex microenvironment of the original organs and mimic the in vivo situation3,4,5,6. Additionally, in monolayer cell cultures, drug treatments could easily destroy/damage all of the cells. Importantly, some of these limitations can be overcome by switching to multilayer three-dimensional (3D) cell cultures7,8. In fact, 3D culture models have been shown to better reflect the cellular structure, layout, and function of cells in primary tissues. These 3D cultures can be formed using a variety of cell lines, similar to a functional organ. Indeed, there are two models of 3D cultures. One model produces spheroids of aggregated cells that form clusters and reorganize them into spheres (scaffold-free models). The second yields organoids, which have a more complex structure and consist of combinations of multiple organ-specific cells, which are considered as a miniature version of organs9,10. Due to this, 3D culture systems represent an innovative technology with many biological and clinical applications. Thus, spheroids and organoids have numerous applications for disease modeling and studies related to regenerative medicine, drug screening, and toxicological studies6,11,12,13,14,15. Carcinogenic spheroids, derived from 3D technology, recreate the morphology and phenotype of relevant cell types, mimic the in vivo tumor microenvironment, and model cell communications and signaling pathways that are operational during tumor development16,17,18. In addition, to improve cancer biology understanding, tumor spheroids/organoids can also be used to identify a potential patient-specific anti-cancer therapy (personalized) and assess its efficacy, toxicity, and long-term effects19,20,21,22. Spheroids have opened prominent opportunities to investigate pathophysiology, disease modeling and drug screening because of their ability to preserve cellular and three-dimensional tissue architecture, the ability to mimic the in vivo situation, and the cell-cell interactions. However, one must also be aware of the limitations of this system, such as the lack of vascular/systemic component, functional immune or nervous system, and the system represents a reductionist approach as compared to animal models. Indeed, in contrast to animal models, 3D structures provide only an approximation of the biology within a human body. Understanding the limitations of the 3D method may help researchers to design more refined and valid processes for producing spheroids that better represent an organ at a larger scale23,24,25.

Cancer is the leading cause of death worldwide, and breast cancer is the most common cancer in women26,27,28. To mimic the complex microenvironment of breast cancer, breast cancer spheroids should be cultured using cells that play a prominent role in breast tumors, i.e., epithelial cells, endothelial cells, fibroblasts, and/or immune cells. Moreover, for a spheroid representing breast cancer, the expression of female hormone receptors (estrogen/progesterone receptors), ability to conserve the patient tumor histological status, and ability to mimic response to therapy should also be considered. Studies have shown that 3D co-culture systems have a cellular organization similar to that of the primary tissue in vivo, have the capability to react in real-time to stimuli, and have functional androgen receptors29,30,31,32. Hence, a similar approach could be useful to mimic a breast tumor in vitro. The purpose of the current protocol is to establish a new method of generating breast cancer spheroids. This method utilizes estrogen receptor-positive MCF-7 cells (an immortalized human cell line of epithelial cells) and vascular endothelial cells (HUVECs) or lymphatic endothelial cells (HMVEC-DNeo) to create a model that mimics or closely reflects the interactions between these cells within a tumor. Although MCF-7 (estrogen-responsive) and endothelial cells have been used to develop spheroids in the present study, other cells such as fibroblasts which represent ~80% of the breast tumor mass, could also be combined in the future to better represent and mimic breast tumor.

There are several methods to form spheroids, such as: 1) the hanging droplet method that employs gravity33,34; 2) the magnetic levitation method that uses magnetic nanoparticles exposed to an external magnet35, and 3) the spheroid microplate method that is performed by seeding cells on low-attachment plates36,37. On the basis of the existing methods, which use only one cell type, the present protocol has been optimized using epithelial and endothelial cells to better mimic the growth conditions of breast cancer tumors in vivo38,39,40,41. This method can be easily achieved in the laboratory at a low cost and with minimal equipment requirements. Based on the need/goals of the lab, different approaches were used to form spheroids and gain relevant cellular material from these spheroids. In this context, for DNA, RNA, or protein analysis, the 3D spheroids are produced by co-culturing endothelial and epithelial cells with the hanging drop method. However, for functional studies, for example, to monitor cell growth after short interfering (siRNA) transfection and/or hormone treatment, the spheroids are generated using U-bottom plates.

The purpose of this technical protocol is to provide a detailed step-by-step description for 1) forming breast cancer multicellular-type spheroids, 2) preparing samples for histological staining, and 3) collection of cells for extraction of RNA, DNA, and proteins. Both the inexpensive hanging drop method and the more expensive U-bottom plates are used to form spheroids. Here, a protocol for preparing (fixing) spheroids for sectioning and subsequent immunostaining with markers to assess cell proliferation, apoptosis, and distribution of epithelial and endothelial cells within a spheroid is provided. Additionally, this protocol shows a complete step-by-step analysis of histological data using ImageJ software. Interpretation of biological data varies depending on the type of experiment and the antibodies used. Sectioning of fixed spheroids and subsequent staining of the sections was performed by a routine pathology lab (Sophistolab: info@sophistolab.ch)

Protokół

1. Cell culture

NOTE: Conduct cell handling under sterile conditions.

  1. Human Umbilical Vein Endothelial Cells (HUVECs) subculture
    1. Coat 75 cm2 flasks with collagen (5 µg/cm2) (rat-tail) overnight (ON) at room temperature (RT) or 2-3 h at 37 °C, rinse with water, and allow the flask to dry.
    2. Grow HUVECs in growth medium (EBM-2, Endothelial Basal Medium-2) supplemented with glutamine (1x = 2 mM), antibiotic-antimycotic solution (AA; 100 µg/mL of streptomycin, 100 µg/mL of penicillin and 0.025 µg/mL of amphotericin B), LSGS (2% v/v FCS, 1 µg/mL of hydrocortisone, 10 ng/mL of human Epidermal Growth Factor, 3 ng/mL of human basic Fibroblast Growth Factor, 10 µg/mL of heparin) and 10% FCS (Fetal Calf Serum) under standard tissue culture conditions (37 °C, 5% CO2). Change the medium every 2 days until the cells reach 70%-80% confluence.
    3. Wash the sub-confluent cultures with 5 mL of HBSS (without Ca2+ and Mg2+) and add 3 mL of trypsin (0.25% diluted in HBSS (without Ca2+ and Mg2+)).
      NOTE: HBSS can also be replaced with PBS.
    4. Incubate the cells at 37 °C for 2 min (microscopically check whether the cells are detached and rounded up) and stop the detaching reaction by adding 6 mL of 10% FCS medium (DMEM-F12 or EBM-2, 10% FCS).
    5. Centrifuge the cell suspension at 250 x g for 5 min at RT and discard the supernatant.
    6. Suspend the cells in growth medium and seed in 75 cm2 flasks or culture dishes.
  2. Michigan Cancer Foundation-7 (MCF-7, human breast adenocarcinoma cells) subculture
    1. Grow human breast cancer cell lines in 75 cm2 flasks in growth medium (DMEM/F12 medium supplemented with a commercial glutamine (1x), antibiotic-antimycotic solution (AA; 100 µg/mL of streptomycin, 100 µg/mL of penicillin and 0.025 µg/mL of amphotericin B), and 10% FCS under standard tissue culture conditions (37 °C, 5% CO2). Change the medium every 2-3 days.
    2. Wash the subconfluent cell cultures (70%-80% confluence) with 5 mL of HBSS (without Ca2+ and Mg2+) and add 3 mL of trypsin (0.5% diluted in HBSS (without Ca2+ and Mg2+) at 37 °C for 5 min (microscopically verify that the cells are detached).
      NOTE: HBSS can also be replaced with PBS.
    3. Add 8 mL of 10% FCS medium to stop the detaching reaction, centrifuge at 250 x g for 5 min at RT and discard the supernatant.
    4. Suspend the pellet in growth medium and plate in 75 cm2 flasks or culture dishes.

2. Spheroid formation

  1. Plate 5 x 105 cells/mL (HUVECs and MCF-7) in a 3.5 cm round dish and culture for 48 h.
  2. Treat or transfect the plated cells for 24 h or as desired.
  3. Optional step performed in 96-well U-bottom plate: Wash the cells with 1 mL of HBSS (Ca2+ and Mg2+) and stain HUVECs using a blue dye (0.5 µg/mL) and MCF-7 cells using a green dye (0.5 µM) diluted in the respective growth medium and place into a 37 °C and 5% CO2 incubator for 30-40 min.
  4. Wash the cells with 1 mL of HBSS (without Ca2+ and Mg2+), add 1 mL of enzymatic detachment reagent (0.25% trypsin for HUVEC and 0.5% trypsin for MCF-7) to each round dish using a P1000 pipette, and then incubate for 2-5 min until the cells are detached.
  5. Collect each cell type separately in a 5 mL round-bottom Polystyrene tube and add 1 mL of 10% FCS medium using a P1000 pipette to stop the enzymatic reaction. Centrifuge the cell suspensions at 250 x g for 5 min.
  6. Carefully aspirate the supernatant and suspend the cells in 1 mL of steroid-free medium (EBM-2, glutamine , antibiotic-antimycotic, 0.4% FCS sf (charcoal-stripped)).
    NOTE: Steroid-free medium can be replaced with normal growth medium.
  7. Use 100 µL of each cell suspension diluted with 10 mL of Diluent II (see Table of Materials) to determine the total cell number and calculate the amount of treatment medium (EBM-2, glutamine, antibiotic-antimycotic, 0.4% FCS sf, vehicle/Treatment) needed to obtain a cell suspension of 5 x 103 cells/mL or 3.4 x 105 cells/mL per cell type.
    1. Cell count
      1. Turn on the machine and flush by pressing the Function and Start buttons (set-up S3), with Diluent II solution in a cell counter vial.
      2. Use set up S4 and press Start to measure the blank with fresh Diluent II solution.
      3. Change the scintillation vial with 100 µL of cell suspension in 10 mL of Diluent II solution and measure the samples: set-up S4 and press Start.
      4. Note the number of cells per mL on the digital display.
        ​NOTE: Cell counting can also be performed using other manual or automatic systems: Hemocytometer gridlines, light-scatter cell-counting technology, electrical impedance, imaging-based systems using cell staining, e.g., trypan blue.
    2. 96-well U-bottom plates
      1. Prepare 5 mL of each cell suspension (5 x 103 cells/mL) and mix them to get a final volume of 10 mL in the ratio of 1:1.
      2. Use a manual repeating pipette to pipette out 100 µL of the cell suspension into each well of the 96-well U-bottom plates.
      3. Place the plate into an incubator under standard tissue culture conditions and check for spheroids formation after 48 h.
      4. Optional step: Take pictures of the spheroids (40x) using a fluorescence stereo microscope.
        NOTE: This method generates 96 spheroids (one spheroid/well) after 48 h (area 1-2 pixel2), and it is usually used for growth studies.
    3. Hanging drop
      1. Mix the cell suspensions (3.4 x 105 cells/mL) in a 1:1 ratio to get a final volume of 2 mL.
      2. Use a P20 pipette to seed the cell mixture in the form of 15 µL drops on the inverted lid of a 10 cm Petri dish.
      3. Invert the lid with the drops and add 5 mL of base medium (EBM-2) at the bottom of the dish to avoid evaporation of the drops.
        ​NOTE: This method generates one spheroid/drop after 48 h. Ensure the drops are sufficiently apart from each other, so that they do not merge while switching the lid. Use more than one 10 cm Petri dish if necessary.

3. Spheroid growth study

  1. Plate 100 µL of HUVECs and MCF-7 cell mixture (5 x 102 cells/well) in 96-well U-bottom plates and place them in the incubator.
  2. 48 h after plating, check for spheroids formation with an inverted microscope (Bright field). Take pictures (at least six pictures per treatment, 40x) at 96 h of culture.
  3. Open the pictures using an image processing software and process the image to 300 pixel/inch and save the image as a tiff file.
  4. Download the ImageJ software and open it. Click on the File option and go to Open.
  5. Convert the areas of interest to saturated black areas in a uniform manner to have a binary image (black and white). For this, select the Image option, choose Type | 8-bit. Now, the image is black and white.
  6. Use the Freehand Selection Tool to draw the border of the spheroids.
  7. To calculate the area of interest and to separate the object or the foreground pixels from the background pixels, use the threshold function: Select the Image option, choose Adjust and click on Threshold.
  8. Now a Threshold pop-up window will open. The top bar indicates the minimum threshold value: set the value at zero. The bottom bar indicates the maximum threshold value: move the bar until the area of interest becomes completely red.
    NOTE: If the color is not red, select Red from the Threshold window.
  9. Go to Analyze | Set Measurements and select Area and Perimeter.
  10. To measure the area of interest, select the Analyze option and use the Analyze Particles tool. In the Analyze Particles window, set the Size to measure (0.00003-Infinity or 3-Infinity, depending on the size of the spheroids), select Summarize and Display Results. Then, click on OK.
  11. The results will show up in a chart. Copy the measurements into a spreadsheet to analyze the data.
    ​NOTE: The area is calculated as the number of total pixels; use the Total Area for calculation.

4. Spheroid immunohistochemistry study

  1. Sample preparation
    1. Plate the cell mixture of HUVECs and MCF-7 (5 x 103 cells/well) in 96-well U-bottom plates in HUVEC growth medium for 96 h.
    2. Collect approximately 50 spheroids into a 1.5 mL tube with a cut tip of a P200 µL pipette; allow them to settle to the bottom of the tube by gravity, and then discard the supernatant.
    3. Fix the spheroids with 500 µL of 4% PFA for 1 h, RT.
    4. Boil 2% Nobel Agar solution in PBS usinga hot plate with a magnetic stirrer for 3-5 min to dissolve the agarose powder completely and cool down to around 60 °C.
    5. Remove the PFA with a P1000 pipette, wash the spheroids with 500 µL of PBS, and allow them to sediment. Discard the supernatant.
    6. Carefully pipette 600 µL of agarose solution into the 1.5 mL tube with spheroids and place the tube immediately in a centrifuge with a horizontal rotor at 177 x g for 2 min.
    7. Add a short string in the middle of the agarose solution to easily remove the plug from the tube.
    8. Solidify agarose plug on ice or at 4 °C. Add 500 µL of PBS into the 1.5 mL tube to avoid drying out of the pellet.
      ​NOTE: The samples are ready for subsequent dehydration, paraffin embedding, sectioning, transfer onto the microscope slides, and IHC staining (performed by Sophistolab).
  2. Image acquisition and processing
    1. Acquire images of spheroid sections using a stereomicroscope.
    2. Save three images for each sample as .jpg files.
    3. Open the ImageJ software. Open the JPEG image, click on File and then on Open.
    4. Select Color and Color Deconvolution in the Image option.
    5. Select H DAB vectors in the Color Deconvolution 1.7 window, and the three images appear.
      NOTE: The three images are: Color 1 represents only the Hematoxylin staining (blue/purple), and Color 2 represents only the DAB staining (brown). Color 3 is not needed for the image analysis with two stainings.
    6. Select the Color 1 window and set the Threshold: go to Image | Adjust and click on Threshold. The Threshold window appears.
    7. Leave the minimum threshold value set at zero and adjust the maximum threshold value to remove the background signal without influencing the true signal. Click on Apply.
      1. Area measurement
        1. Click on Analyze, choose Set Measurement. Select Area, Mean grey value, and Min and Max grey value and click on OK to confirm.
        2. Measure the size of the nucleus using the Analyze option, and then select Measure.
          NOTE: The Results window gives the name of the image (Label), size of the image (Area), the average pixel intensity of the IHC image (Mean), and the minimum and maximum gray values (Min, Max). Use the Mean value for calculation.
        3. Repeat steps 4.2.6-4.7.1.2 for the Color 2 (and Color 3 if there is dual staining) window.
      2. Cell quantification
        1. Click on Process, choose the option Binary and select Watershed to separate cells.
        2. Go to Analyze and click on Analyze Particles. On the Analyze Particles pop-up window, set the size of the cells to exclude unspecific particles. Next, on the Show option, click on the drop-down box and select Outlines. Then, click on OK.
        3. Repeat steps 4.2.6-4.2.7 and cell quantification steps (section 4.2.7.2) for the Color 2 (and Color 3 if there is dual staining) window.
          ​NOTE: Now three windows will appear: 1) Summary results (number of cells counted, total area, average size, % of area and mean), 2) Results (respective to the cells that are counted), and 3) Drawing window (depicted image of the cells that are counted).

5. Live/dead staining in Spheroids

  1. Prepare 4 mM stock solutions of Calcein AM in DMSO (stains live cells green) and 2 mM of Ethidium homodimer in DMSO (stains dead cells red) in 1.5 mL tubes.
  2. Calculate the amount of solution necessary to add 10 µL/well of a mixture of Calcein AM and Ethidium homodimer diluted 1:50 in EBM-2.
  3. Add the staining mixture to the spheroids and place the plate in the incubator under standard tissue culture conditions for 30-60 min.
  4. Acquire pictures (40x) using the Fluorescence stereo microscope.

6. Spheroid's protein and RNA isolation

  1. Prepare cell suspensions at 3.4 x 105 cells/mL per cell type, mix them at a ratio of 1:1, and seed the cell mixture using a P20 pipette in the form of 15 µL drops on the lid of a 10 cm Petri dish. Invert the lid and place it in the incubator under standard tissue culture conditions for 96 h.
  2. Use 5-6 mL of PBS to collect spheroids in a 15 mL round-bottom Polystyrene tube using 5 mL sterile polystyrene pipettes.
    NOTE: It is also possible to use 15 mL conical tubes.
  3. Centrifuge the spheroids suspension at 250 x g for 5 min, and then carefully aspirate the supernatant.
  4. Add 500 µL of trypsin (100%) and pipette up and down using a P1000 pipette for 30 s to disaggregate spheroids, achieving a cell suspension.
  5. Neutralize trypsin with 10% FCS medium, centrifuge the tubes at 250 x g for 5 min, and aspirate the supernatant.
  6. According to the manufacturer's protocol (specific kit for DNA-free RNA isolation), use 300 µL of RNA lysis buffer to lyse the cell pellet.
    NOTE: Lysis buffer can also be directly added to the intact spheroids (no trypsin step required) to extract RNA. Add 300 µL of the lysis buffer to the pelleted spheroids, place tubes in ice, and triturate 10 times using a P200 pipette. Subsequently, isolate RNA as above. Spheroid dissociation may be needed if RNA recovery is low due to matrix interference.
  7. Alternatively, use 70 µL of the Lysis buffer for protein isolation. Homogenize for 2-4 s by sonication and determine the protein concentration according to the manufacturer's protocol using a BCA Assay Kit.
    NOTE: For protein isolation, pelleted spheroids can be lysed directly in lysis buffer followed by sonication. Use 25 µg of total protein to perform western blot.

Wyniki

The spheroids model using epithelial and endothelial co-cultures is required to closely mimic in vivo conditions of breast tumors for in vitro experiments. The scheme in Figure 1 depicts the protocol to form spheroids with breast cancer epithelial cells and vascular or lymphatic endothelial cells (Figure 1). Each cell type is seeded separately in a 3.5 cm round dish and treated with growth stimulators/inhibitors or transfected with oligonucleot...

Dyskusje

Compared to 2D cell cultures, revolutionary 3D spheroid culture technology is a better and more powerful tool to reconstruct an organ's microenvironment, cell-cell interactions, and drug responses in vitro. This is the first protocol describing the formation of spheroids from multicellular (epithelial and endothelial) cell lines for breast cancer research. This protocol ensures spheroidal 3D growth of spheroids for up to 5 days, and spheroids can be examined after paraffin embedding, sectioning, and histolog...

Ujawnienia

The authors have nothing to disclose.

Podziękowania

This research was supported by Cancer Research Foundation / Swiss Cancer League grant KFS-4125-02-2017 to RKD and National Institute of Health grant DK079307 to EKJ.

Materiały

NameCompanyCatalog NumberComments
100 mm × 20 mm tissue-culture treated culture dishesCorningCLS430167
149MULTI0C1
35 x 10 mm Tissue Culture DishFalcon353001
5 mL Serological Pipet, Polystyrene, Individually Packed, SterileFalcon357543
Adobe PhotoshopVersion: 13.0.1 (64-bit)
Antibiotic Antimycotic Solution (100×)Sigma-AldrichA5955
Calcein-AMSigma-Aldrich17783
CD31 (cluster of differentiation 31)Cell Marque131M-95monoclonal mouse ab clone JC70
CellTracker Green CMFDA (5-chloromethylfluorescein diacetate)InvitrogenC7025
CK8/18 (cytokeratins 8 and 18)DBSMob189-05monoclonal mouse ab, clone 5D3
CKX41 Inverted MicroscopeOlympus Life ScienceOlympus DP27 digital camera
Cleaved Caspase 3Cell Signaling9661Lpolyclonal rabbit ab
Collagen (rat tail)Roche11 179 179 001
Coulter ISOTON II DiluentBeckman Coulter844 80 11Diluent II
Coulter Z1, Cell CounterCoulter Electronics, Luton, UK
Dehydrated Culture Media: Noble AgarBD DifcoBD 214220
Dimethyl sulfoxide (DMSO)Sigma-AldrichD2650
Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 HamSigma-AldrichD6434
EBM-2 Endothelial Cell Growth Basal Medium-2Lonza190860
Ethidium homodimerSigma-Aldrich46043
Fetal Calf Serum (FCS)Thermo Fisher ScientificSH30070
Fetal Calf Serum Charcoal Stripped (FCS sf)Thermo Fisher ScientificSH3006803
Fluorescence stereo microscopes Leica M205 FALeica Microsystems
GlutaMAX Supplement (100x)Gibco35050038
HBSS, no calcium, no magnesium, no phenol redGibco14175053
Hoechst 33342Life TechnologiesH3570
HUVEC – Human Umbilical Vein Endothelial CellsLonzaCC-2517
ImageJNational Institute of Health, USAWayne Rasband
Version: 1.52a (64-bit)
Ki67Cell Marque275R-16monoclonal rabbit ab, clone SP6
Leica Histocore Multicut Rotary Microtome149MULTI0C1
Low Serum Growth Supplement Kit (LSGS Kit)GibcoS003K
MCF-7 cells – human breast adenocarcinoma cell lineClinic for Gynecology, University Hospital ZurichProvived from Dr Andrè Fedier obtained from ATCC
Nunclon Sphera 96U-well plateThermo Fisher Scientific174925
Paraformaldehyde (PFA)Sigma-AldrichP6148
Phosphate-buffered saline (PBS) 1xGibco10010015
Pierce BCA Protein Assay KitThermo Scientific23225
Protein Lysis BufferCell Signaling, Danvers, USA9803
Quick-RNA Miniprep KitZymo ResearchR1055
RNA Lysis BufferZymo ResearchR1060-1-100Contents in Quick-RNA Miniprep Kit
Rotina 46R CentrifugeHettich
Round-Bottom Polystyrene Tubes, 5 mLFalcon352003
SonicatorBandelin electronics, Berlin, DE
Tecan Infinite series M200 microplate readerTecan, Salzburg, AU
Tissue Culture Flasks 75TPP90076
Trypsin-EDTA solutionSigma-AldrichT3924

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