$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
NOTE: An overview of the entire MEMA process, including estimated time, is outlined in the flow diagram shown in Figure 1. This protocol details the fabrication of MEMAs in 8-well plates. The protocol may be adapted for other plates or slides.
1. Preparation of Protein, Diluent, and Staining Buffers
- Equilibrate vials of ECMs, ligands, and cytokines to room temperature (RT) and briefly centrifuge. Add the appropriate volume of the appropriate RT buffer as indicated on the product data sheet. Follow manufacturer’s recommendation for stock concentrations.
NOTE: A full list of the ligands and ECMs with their stock and final concentrations are provided in Table 1 and Table 2. Both ligands and ECMs are typically used at the highest concentration of the range recommended by the manufacturer that elicits a biological effect in standard 2 day culture assays. Handle proteins gently and in biosafety cabinets under laminar flow to avoid contamination.
- Incubate vials with gentle rocking at RT for 1 h. Do not vortex proteins as this can cause them to denature.
- Aliquot proteins for long term storage so that all aliquots are single use only to avoid degradation with repeated freeze/thaw cycles. Store lyophilized proteins at -80 °C (unless otherwise specified) until needed. Take care to collect all metadata for future reference, such as: (i) protein name, (ii) date prepared, (iii) lot/batch number, (iv) supplier, (v) catalog number, (vi) concentration, (vii) volume, and (viii) preparer.
- Prepare diluent buffer containing 20% (v/v) glycerol, 10 mM EDTA, 200 mM Tris-HCl, pH 7.2, and filter sterilize. Keep this buffer sterile and store at RT.
- Prepare staining buffer containing 2% (w/v) BSA, 1 mM MgCl2, and 0.02% NaN3 in phosphate-buffered saline (PBS). Filter and store at 4 °C.
2. Preparation of an ECM Source Plate
- Remove aliquoted stocks of ECM proteins to be printed and thaw on ice. Record all lot numbers for metadata tracking.
- Flick tubes of thawed proteins gently to ensure proper resuspension and spin down in a centrifuge.
- Make ECM print mixtures (EPMs) and a fluorescent fiducial to be used by a liquid handling robot that will create the randomized 384-well source plates.
NOTE: The 384-well source plates will be used by a touch pin array printer to create the printed arrays in 8 well plates.
- Label 1.5 mL microcentrifuge tubes for each EPM and the fiducial.
- Prepare each EPM by combining 125 µL of diluent buffer (see step 1.4) with the appropriate volume of ECM stock and bring the mixture up to a total volume of 250 µL with PBS. The final concentrations in each EPM tube will be 1x ECM protein, 5 mM EDTA, 10% glycerol, and 100 mM Tris.
- Prepare a fluorescent fiducial by dissolving it in the appropriate buffer specified by the manufacturer and transfer 250 µL to a labelled fiducial tube.
3. Creation of the Source Plate Using a Liquid Handler
- Design a 384-well plate layout that randomizes the positions of the ECMs and is optimized for the array printer pin head being used. Design the placement of the fiducial so that it will be printed in the row 1, column 1 position of each well to assist in array orientation.
NOTE: A total of 14−15 replicates of each ECM are used to ensure robust data. Include additional replicates of collagen or another ECM that yields robust attachment for assessment of uniformity of binding. The layout may need to utilize multiple 384-well plates depending on the number of ECMs of interest.
- Transfer EPM tubes to a liquid handler, keeping tubes at 4 °C either with a cooled tube rack or by using a liquid handling robot located in a cold room.
- Using the liquid handler’s software, run a program to transfer 15 µL of each EPM and the fiducial to the predesignated wells within the 384-well source plate(s).
- Pipet PBS into any unused wells to increase humidity and guard against desiccation during the printing process.
NOTE: See Figure 2 for an example of a 384-well source plate set that is optimized for a 4 x 7 pin head and includes a collagen I block and PBS.
- Seal plate(s) and keep at 4 °C until ready to print.
4. Printing MEMAs Using an Array Printing Robot
NOTE: The following part of the protocol specifically describes the preparation and use of MEMA to investigate the impact of different microenvironment proteins on the growth and proliferation of MCF7 cells. However, the protocol can easily be adapted to use different ligands, ECMs, and cells to study other cell lines and endpoints of interest.
- Using a touch pin printer, print EPMs and fiducial spots into 8 well plates. Print multiple replicates of each ECM condition to ensure reproducibility.
NOTE: Other plate formats or slides can be used for printing, but buffer optimization may be required to achieve optimal spot formation.
- Print the ECMs for the MEMA using 350 µm diameter pins arranged in a 4 x 7 print head configuration. Print the arrays in the 8-well plates as 20 columns by 35 rows, for a total of ~700 spots. Larger arrays are possible in these plates but come with a trade-off of increased edge effects in both cell binding and staining.
- After printing, store plates in a desiccator for a minimum of 3 days prior to use.
5. Creation of Ligand Treatment Plates
- Design a 96-well plate layout including ligands of interest. To facilitate treatment of many MEMA plates at once, design this plate with spacing that allows for the use of a multi-channel pipet with 4 spaced tips to transfer liquids between the wells of 8-well MEMAs and a 96-well plate.
NOTE: In this protocol, the full set of ligands listed in Table 2 are utilized.
- Thaw ligands on ice. Briefly flick and spin down each tube.
- Dilute ligands to a 200x working stock using the manufacturer’s recommended buffer (typically PBS).
- Pipet 10 µL of each 200x ligand stock into the corresponding well within the 96-well plate.
- Seal and store plates at -20 °C.
NOTE: Make ligand treatment plates in batches, capturing all metadata for downstream analysis.
6. Culturing Cells on MEMAs
- Block MEMAs for 20 min with 2 mL per well of non-fouling blocking buffer containing 1% non-fouling blocking agent (Table of Materials) in double-distilled water (ddH2O).
- Aspirate blocking buffer and triple rinse wells with PBS. To prevent desiccation, leave final volume of PBS in wells until ready for cell plating.
NOTE: It is extremely helpful to have two bench workers for cell culture steps on MEMAs. One bench worker can perform aspiration steps, while the second performs addition steps. It is recommended to use a 1 mL multichannel pipet with tips spaced to match the 8-well plate for pipetting and a Y-splitter with two Pasteur pipettes to aspirate multiple wells at once.
- Seed 2 x 105 MCF7 cells per well in 2 mL of Dulbecco’s modified Eagle’s medium (DMEM) medium containing 10% fetal bovine serum (FBS).
NOTE: Prior to a full MEMA experiment, perform a cell titration experiment to optimize cell numbers such that MEMA spots have high cell numbers (but are not confluent) at the end of the desired experimental duration.
- After 2−18 h of adhesion, aspirate medium and replace with 2 mL of reduced-growth medium (DMEM with 0.1% FBS).
NOTE: Reduced serum (e.g., 0.1% FBS) or growth factor-depleted conditions can be used at this time to isolate the stimulatory impact of specific ligands.
- Thaw a ligand treatment plate on ice. Centrifuge thawed plate at 200 x g for 1 min.
- Transfer 200 µL of medium from each well in the culture plate to the appropriate well in the treatment plate. Pipet up and down to mix ligand volume with medium and transfer this mixture back to the appropriate well in the MEMA plate.
- Lightly rock by hand and return MEMA plates to the incubator. Culture for the duration of the experiment in the presence of the ligand/ECM combination at 37 °C and 5% CO2.
NOTE: A typical MEMA experiment runs for 72 h; longer duration experiments may require replacement of medium and re-treatment with ligand.
- Pulse MEMA wells at 71 h with 100x 5-ethynyl-2’-deoxyuridine (EdU) for a final concentration of 10 µM. Incubate in experimental conditions with EdU for 1 h at 37 °C and 5% CO2.
NOTE: Other live cell treatments may also be used at this time.
7. Fixing and Staining MEMAs
- After 72 h and any live cell treatments, aspirate wells. Fix MEMAs in 2 mL per well of 2% paraformaldehyde (PFA) for 15 min at RT.
- Aspirate PFA. Permeabilize with 2 mL per well of 0.1% nonionic surfactant for 15 min.
- Aspirate the nonionic surfactant and wash with 2 mL per well of PBS. Aspirate PBS. Wash with 2 mL of PBS with 0.05% polysorbate 20 (PBS-T).
NOTE: The MEMA surface is hydrophobic, and failure to wash with PBS-T before stain and antibody incubation will result in the formation of voids in wells during incubation steps and give rise to staining artifacts.
- Aspirate PBS-T. Add EdU detection reaction reagents. Incubate for 1 h at RT, rocking and protected from light. After 1 h incubation, quench reaction with the provided commercial quench buffer.
NOTE: EdU detection and staining/antibody steps may be performed in 1.5 mL per well to reduce cost.
- Aspirate the quench buffer and wash with PBS-T prior to incubating with stains or antibodies.
- Incubate MEMA wells with antibodies against histone H3K9me3 (1:1,000) and fibrillarin (1:400) in staining buffer containing 2% (w/v) bovine serum albumin (BSA), 1 mM MgCl2 and 0.02% NaN3 overnight at 4 °C.
NOTE: Perform antibody titrations to determine optimal concentrations prior to using them on a full MEMA set.
- Following primary antibody or stain incubation, wash wells 2x with PBS and once with PBS-T.
- Add secondary antibodies (donkey anti-mouse IgG and donkey anti-rabbit IgG, both 1:300) and 0.5 µg/mL 4′ 6‐diamidino‐2‐phenylindole (DAPI). Incubate for 1 h at RT in the dark.
- Wash wells 2x with 2 mL per well of PBS, leaving them in the final 2 mL PBS.
- Proceed to imaging or store stained MEMAs for later imaging in PBS at 4 °C protected from light.
8. Imaging of MEMAs
- Image MEMA on an automated imaging system with appropriate fluorescent detection channels.
- Output resulting image data to an image management system. Segment cells and calculate intensity levels using CellProfiler8.
9. Data Analysis
NOTE: Data analysis consists of normalization, variation correction, and summarization of the raw CellProfiler derived data. In this instance, the R-environment with custom code is used to perform all the steps. However, any statistical environment or software program can be utilized to perform the equivalent actions. An example of the open source custom code for the R environment for analysis is available at: https://www.synapse.org/#!Synapse:syn2862345/wiki/72486.
- Preprocess and normalize the segmented image data.
- Determine spot cell count using the DAPI stained nuclei.
- Auto-gate EdU intensity to label cells as EdU+. Measure proliferation using the proportion of EdU+ cells in each spot.
- Median summarize cytoplasmic stains and nuclear morphology measurements on the spot level.
- Perform removal of unwanted variation (RUV) normalization on the data to improve data quality9.
NOTE: This approach is applied to each intensity and morphology signal independently as a matrix with arrays using the rows and spots as the columns as described previously9.
- Apply bivariate loess normalization to the RUV normalized residuals using the array row and array column as the independent variables to correct for spatial or intensity related effects.
- Once normalization is completed, median summarize the replicates for each microenvironment condition for reporting and further analysis.