The described platform is intended to screen up to 14 antibodies at a time against a single protein target and can be adapted to test hundreds. All timings listed below do not include cell culture time. The cell culture requirement is sufficient to screen, in parallel, 14 antibodies directed against the same protein target, in 3 applications.
Procedure 1: Antibody screening by WB ● Timing 2 Days
1A) Protein extraction
Option 1: Cell lysate preparation for WB – intracellular protein ● Timing 1.5 hrs (Day 1)
i. Grow 2 x 150 mm dishes of parental cells and 2 x 150 mm dishes of KO cells to 80% confluence in complete medium (see the section “Cell preparation for Antibody Screening” for confluence instructions).
ii. Place the culture dish on ice, remove and discard medium.
iii. Wash the adherent cells three times with ~10 ml of ice-cold PBS 1x. Ensure total removal of PBS between washes using, for example, a vacuum.
iv. After the last wash, add 1.0 ml of RIPA lysis buffer supplemented with protease inhibitor cocktail mix to each 150 mm dish.
v. Use cell scrapers to detach adherent cells.
vi. Collect and pool the cell lysates from the same condition together into a 5 ml tube.
vii. Sonicate both cell lysates 3 x 5 sec at 40% amplitude.
viii. Rock for 30 min at 4 °C.
ix. Centrifuge at ~110,000 x g for 15 min at 4°C using a refrigerated ultracentrifuge.
CRITICAL STEP Ultra-speed centrifugation pellets insoluble contaminants that would otherwise adhere to the beads-antibody conjugate and subsequently interfere with the detection of the captured protein in WB. While maximum speeds achievable with a table-top centrifuge (15,000 - 20,000 x g) are commonly employed to clear lysates, we have observed that such centrifugation does not adequately remove insoluble particles.
x. Gently remove the tubes from the rotor and place them on ice.
xi. Transfer supernatants to fresh 1.5 ml microtubes kept on ice. Discard pellets.
PAUSE POINT Aliquots of parental and KO cell lysates can be stored at -20 °C for six months and at -80 °C for a year.
Option 2: Culture medium collection for WB – secreted protein ● Timing 1.5 hrs (Day 1)
i. Grow 3 x 150 mm dishes of parental cells and 3 x 150 mm dishes of KO cells to 80% confluence in complete medium.
ii. Wash all dishes 3x with sterile warm PBS 1x under a laminar flow cell culture hood.
iii. Add 20 ml of warm serum-free medium to each 150 mm plate.
CRITICAL STEP A serum-free medium is used to avoid contaminating the pool of secreted cellular proteins with highly abundant exogenous proteins present in bovine serum.
iv. Incubate plates in an incubator at 37°C, 5% CO2 for 18 hrs.
v. Collect media in 50 ml conical tubes on ice.
vi. Centrifuge the 50 ml tubes at 500 x g for 10 min at 4 °C to eliminate cells and large contaminants.
vii. Transfer the supernatants to new 50 ml conical tubes and centrifuge at 4,000 x g for 10 min at 4 °C to eliminate small contaminants.
viii. Transfer the supernatant to new 50 ml conical tubes on ice.
ix. Add 15 ml of cleared medium to each 15 ml centrifugal filter unit.
CRITICAL STEP Selection of the appropriate nominal molecular weight limit depends on the target protein. For example, the 10 kDa cutoff filter units can be used for proteins with a molecular weight higher than 10 kDa.
x. Centrifuge at 4,000 x g for 30 min at 4 °C. In each filter unit, a volume of ~500 µl of medium remains after centrifugation, resulting in a ~30-fold concentrated medium.
CRITICAL STEP If the protein concentration is too low and further centrifugation is required, 0.5 ml centrifugal filter units can be used.
xi. Collect the concentrated medium into 1.5 ml microtubes on ice.
xii. Add the corresponding volume of 100x protease inhibitor cocktail mix for a final concentration of 1x.
PAUSE POINT Aliquots of concentrated media can be stored at -20 °C for one year.
1B) Sample preparation for WB ● Timing 1.5 hrs (Day 1)
i. Measure protein concentration using a BCA protein assay kit for lysates (intracellular protein) and a Bradford reagent for media (secreted protein).
CRITICAL STEP Precise monitoring of protein concentration is key to interpret antibody specificity by WB. Measurement of protein concentration in triplicate allows more precise and reproducible quantification for both the BCA and Bradford assay.
ii. Adjust the protein concentrations so that equal amounts of protein are loaded in each lane.
CRITICAL STEP Antibody signal (band intensity) follows a linear and proportional relationship with the protein concentration used to test every antibody/ target pair33. To reach this linear range of detection, we load a defined amount of protein depending on the putative abundance of the target in the chosen parental cell line. To do so, we first search the protein target in PAXdb34 (RRID:SCR_018910, pax-db.org) to determine protein abundance in the selected cell line. PaxDB provides protein abundance in parts per million, or ppm. We use 50 µg of lysate or medium for low abundance proteins between 10 and 1000 ppm and 20 µg of lysate or medium for high abundance proteins with over 1000 ppm. Even with these considerations, primary antibody titration may be required later.
1C) Protein electrophoresis ● Timing 2 hrs (Day 1)
i. Select the appropriate type of polyacrylamide gels based on the target protein size. 12-well midi precast gels running under denaturing conditions are suggested here (Table 1).
CRITICAL STEP Various gel chemistries are commercially available with various advantages and disadvantages. While larger proteins are often considered harder to detect, we found that the commonly used Tris-Glycine (TG) gels are suitable for proteins up to 500 kDa. As an example, Plectin (PLEC), a large protein expected at 532 kDa, was detected using a KO-validated antibody on a TG (Fig. 8a, left WB), a Bis-Tris (BT) gel (Fig.8a, middle WB) combined with the MOPS buffer and on a Tris-Acetate (TA) gel (Fig.8a, right WB). BT and TA gels provided a better separation and resolution of PLEC than the TG. However, the TG gel was still revealed to be suitable for detecting PLEC and is chosen for standardization purposes in this large effort on antibody characterization as it covers the largest molecular weight spectrum. TG and BT gels were transferred in 1x Tris/Glycine buffer as [CL1] detailed in Table 2. TA gel was transferred following the same parameters as the BT gel. All transfer conditions were inspired from manufacturers’ recommendations and then further optimized.
On the other hand, standard transfer conditions from the TG gel were not suitable for transferring small proteins successfully (Fig. 8b, left WB). Therefore, BT gels combined with MES buffer are required for our procedures as this chemistry was highly efficient at separating and resolving the small 9 kDa protein, FCER1G (Fig. 8b, right WB).
ii. Prepare a master mix for both parental and KO cell lysates at the same protein concentration to facilitate sample loading into gels.
iii. Add loading sample buffer to a final concentration of 1x. Depending on the gel chemistry chosen above, samples must be prepared with the compatible type of loading sample buffer (Table 1). Similarly, prepare a master mix of molecular weight markers; volume can be increased with RIPA buffer to match the volume of cell lysate master mix. Chose the appropriate type of molecular weight marker for your protein. Two types of molecular weight markers are listed in Table 1; the BLUelf Prestained Protein Ladder covers proteins from 5 – 245 kDa while the HiMark Pre-Stained High Molecular Weight Protein Standard is suggested for proteins above 245 kDa.
iv. Heat the master mixes of protein samples and molecular weight markers for 10 min at 65°C in a heat block to help dissolve the SDS and/or glycerol in the loading sample buffer, which facilitates the loading into the gel.
CRITICAL STEP For some proteins, boiling samples can create artifacts. The G protein-coupled receptor S1PR1 runs as two major bands (~44, 48 kDa) with additional minor bands detected below and above both major bands (Fig. 7, left panel). Boiling the samples led to an intense artifactual smear above 245 kDa and a reduction of the signal at ~44 and 48 kDa (Fig. 7, right panel).
v. Pulse spin the samples and molecular weight master mixes using a microcentrifuge, and load samples into a 12-well polyacrylamide gels in the order suggested in Fig. S1a. A total of 4 antibodies can be tested on each 12-well gel. To test 14 antibodies, 4 x 12-well gels are required.
vii. Follow the conditions recommended by the manufacturer to run the gel using the appropriate running buffer (Table 1), until the dye front reaches ~3 mm from the bottom.
1D) Protein transfer to nitrocellulose membrane ● Timing 1.5 hrs (Day 1)
i. Transfer proteins from the gel to a membrane; nitrocellulose membranes are used here. The Bio-Rad criterion blotter is the wet transfer system employed. Transfer conditions are detailed in Table 2.
ii. Take the membrane out of the sandwich and wash twice with deionized water in a container.
iii. Stain all transferred proteins by covering the membranes with the Ponceau S solution.
iv. Incubate for 1 min; Ponceau S staining solution can be reused several times.
v. Wash off excess Ponceau S with deionized water until the area of membranes not covered by proteins becomes white.
vi. Dry the Ponceau S-stained membranes on Whatman filter paper.
vii. Label membranes properly with a suitable smudge proof pen.
viii. Scan membranes using a regular paper scanner.
ix. Trim membrane strips containing each a molecular weight marker and a WT/KO lysate pair.
PAUSE POINT Dried membranes can be stored at room temperature for months.
1E) Blocking and primary antibody incubation ● Timing 1 hr and O/N (Day 1 and 2)
i. Rehydrate membrane strips and remove Ponceau S staining by incubating the membrane in TBST 1x for 5 min.
ii. Block the membrane with the WB blocking solution for 1 hour at room temperature.
iii. During the blocking step, prepare the primary antibody solution in WB blocking solution.
CRITICAL STEP The initial concentration of primary antibody tested is in accordance with the manufacturer's recommendations. Antibodies are titrated only when the signal falls outside of the linear dynamic range.
iv. Incubate each membrane strip with the corresponding primary antibody dilution. A container or a resealable flat plastic bag can be used for the incubation.
v. Rock overnight at 4°C.
1F) Secondary antibody incubation and signal detection ● Timing 3 hrs (Day 2)
i. Discard antibody solution and wash membrane strips in a container with TBST 1x for 10 min under constant rocking. Repeat twice more for a total of 3 washes.
ii. During washes, dilute the corresponding HRP-conjugated secondary antibody in WB blocking solution to a concentration of 0.2 - 0.5 µg/ml.
iii. Rock membrane blot strips with the corresponding HRP-conjugated secondary antibody in a container or a sealed plastic bag for 1 hr at room temperature.
iv. Wash 3 x times in TBST 1x for 10 min with constant rocking.
v. After the last wash, place membrane strips on a clean surface and incubate with the peroxidase substrate for 1 min, then remove excess.
vi. Place membrane strips in a chemiluminescence imaging system and follow the manufacturer’s guidelines for signal detection. The iBright imaging system is suggested here.
CRITICAL STEP Signal strength varies depending on the antibody and its concentration. Several exposures must be typically taken to observe the bands of interest at intensities comparable between the different antibodies.
vii. Name images properly and export the different exposures for later figure preparation.
viii. Analyze the band pattern to assess antibody specificity (Fig. 2a).
CRITICAL STEP Identification of a cell line with confirmed endogenous expression of the target, assessed using a KO-validated antibody, is essential to proceed with IP-WB and IF procedures.
PAUSE POINT The next procedures can be started at any time after screening antibodies by WB was successful.
Procedure 2: Antibody screening by IP ● Timing 3 days
2A) Protein extraction
Option 1: Cell lysate preparation for IP – intracellular protein ● Timing 1.5 hrs (Day 1)
i. Grow a 150 mm dish of parental cells to generate enough lysate for two IPs (7 dishes if 14 antibodies are tested).
ii. Place each culture dish on ice and discard medium.
iii. Wash the adherent cells three times with ~10 ml of ice-cold PBS 1x. Ensure total removal of PBS between washes with, for example, a vacuum.
iv. After the last wash, add 1.0 ml of ice-cold IP lysis buffer supplemented with 1x protease inhibitor cocktail mix to each 150 mm dish.
v. Use cell scrapers to gently detach adherent cells from the petri dish.
vi. Collect and pool parental cell lysates into a 5 or 15 ml tube, depending on the number of dishes used.
vii. Rock cell lysates for 30 min at 4 °C.
viii. Centrifuge at ~110,000 x g for 15 min at 4°C (as with WB).
ix. Gently remove the tubes from the rotor and place them on ice.
x. Pool the supernatants into the same tube kept on ice. Discard pellets.
CRITICAL STEP Freshly prepared lysates must be used for the IP experiment. Freezing the protein sample might affect the epitope to be recognized by the antibodies tested.
Option 2: Culture media collection for IP – secreted protein ● Timing 1.5 hrs (Day 1)
i. Grow a 150 mm dish of parental cells for each IP (15 dishes if 14 antibodies are tested).
ii. As described in the WB procedure 1A option 2, wash and grow the cells without serum for 18 h, then collect and concentrate media.
iii. Combine all concentrated media from parental cells into the appropriate tube.
iv. Add the corresponding volume of 100x protease inhibitor cocktail mix for a final concentration of 1x.
CRITICAL STEP Freshly collected and concentrated media must be used for the IP experiment.
2B) Sample preparation for IP ● Timing 1.5 hrs (Day 1)
i. Measure protein concentration using a BCA protein assay kit (lysate) or Bradford reagent (medium).
ii. For lysates, adjust protein concentration to 2.0 mg/ml with IP lysis buffer. 1 mg (500 µl at 2.0 mg/ml) is used later for each IP.
For concentrated media, the concentration is usually at ~1 mg/ml. 500 µg (500 µl at 1.0 mg/ml) is used later for each IP.
iii. Save enough lysate for starting material sample.
CRITICAL STEP 4% starting material is suggested here. 20 µl of lysate at 2.0 mg/ml (40 µg) or 20 µl of medium at 1.0 mg/ml (20 µg) are kept aside to run side-by-side with each IP. For 14 antibodies, save ~300 µl for starting material.
2C) Antibody-beads conjugation ● Timing 1.5 hrs (Day 1)
i. Select the appropriate type of magnetic beads for each antibody (Table 3)35.
ii. Resuspend the beads slurry.
iii. Label enough microtubes (one microtube per antibody to be tested) and add 30 µl of magnetic beads slurry to 1.0 ml of IP lysis buffer.
iv. Add 2.0 µg of the corresponding primary antibody.
TROUBLESHOOTING When the antibody concentration is not provided by the manufacturer, follow their volume recommendation for IP, if applicable. If not, test a certain volume of the antibody and keep a record of the volume tested.
v. Maintain constant agitation on a rotating mixer for 1 hr at 4°C.
vi. Place tubes on the DynaMag-2 magnet and allow 15 sec for the beads to attach to the magnet.
vii. Vacuum out the buffer to remove unbound antibodies.
viii. Add 1.0 ml of IP lysis buffer and allow release of the beads by taking the tube off the magnet.
ix. Wash the beads by inverting the tube multiple times to resuspend.
x. Repeat to wash a second time and remove the excess unbound antibodies.
CRITICAL STEP Do not let the beads dry out at any step.
2D) Immunoprecipitation ● Timing 2 hrs (Day 1)
i. Remove buffer from the antibody-bead conjugate using the magnet.
ii. Add 500 µl of lysate at 2.0 mg/ml or 500 µl of medium at 1.0 mg/ml to each tube of antibody-conjugated beads.
iii. Incubate antibody-beads conjugates with the lysate for 1 hr at 4°C with constant agitation on a rotating mixer.
iv. Place each microtube on DynaMag-2 and allow at least 15 sec for the beads to converge toward the magnet.
v. From each tube, collect 20 µl of samples which represent the unbound fraction (proteins that did not bind to the antibody-bead conjugate).
vi. Pipet each unbound fraction in a labelled microtube, set aside on ice.
vii. Vacuum out any remaining samples from each tube on the DynaMag-2.
viii. Wash the magnetic beads 3x in 1 ml IP lysis buffer supplemented with protease inhibitor cocktail mix.
ix. After last wash, elute with 30 µl of the appropriate loading sample buffer diluted to 1x in IP lysis buffer.
x. Add loading sample buffer to the starting material and unbound fractions. Similarly, prepare molecular weight marker sample (volumes should be completed with IP buffer to match sample volumes).
CRITICAL STEP The final concentration of loading sample buffer must be identical in all samples (usually 1x).
xi. Heat all samples for 10 minutes at 65°C in a heat block.
PAUSE POINT Samples can be left at room temperature for one day or stored at -20°C for several weeks.
2E) WB assessment of antibody performance by IP
● Timing 4.5 hrs (Day 2), O/N and 3 hrs (day 3)
i. Select the same polyacrylamide gel as selected for the WB screening (Table 1).
ii. Load samples on 12-well polyacrylamide gels in the order suggested in Fig. S1b. A total of 3 antibodies can be evaluated from a single 12-well gel. To test 14 antibodies, 5 x 12-well gels are required.
iii. The following WB is done using KO-validated antibodies selected from the WB screening (procedure 1).
CRITICAL STEP Immunoglobulins from the antibodies used in the IP are eluted with the loading sample buffer containing reducing agents. For rabbit polyclonal antibodies, the heavy chain runs at 50 kDa, and the light chain at 25 kDa. For other types of antibodies, the molecular weight of these chains might vary. The same primary antibody is ideally used in WB to assess the performance of all antibodies tested by IP.
TROUBLESHOOTING When the proteins of interest have a molecular weight close to that of immunoglobulin chains, the antibody used in WB may cross-react with these chains, masking the protein signal. These cross-reactions can also create noise elsewhere in the IP pathway (Fig. 9a, case 1 and Fig. 9b, case 4). Two options are proposed to solve this problem: 1) use a secondary detection system that should not react with unstructured immunoglobulins to avoid these cross-reactivities (Fig. 9a, case 2 and Fig. 9b, case 5) or 2) where possible, choose a primary KO-validated antibody raised in a different host than the antibody used in IP, ideally allowing minimal detection of immunoglobulins from different species (Fig. 9a, case 3 and Fig. 9b, case 6).
iv. Perform the WB as detailed in procedure 1E, F.
v. Analyse the data by comparing the WB signal in the starting material, unbound fraction and immunoprecipitate (Fig. 5).
Procedure 3: Antibody screening by IF ● Timing 2 d
3A) Prepare parental/KO mosaic in 96-wells for IF ● Timing 2 hrs (Day1)
i. Coat each well of a 96-well, clear flat bottom plate with 100 µl of poly-L-lysine working solution. The suggested 96-well plate, Revvity cat. no 6055300, is compatible with cell imaging and most high-content imaging systems.
ii. Incubate one hour at room temperature.
iii. Wash each well twice with 100 µl of sterile water.
iv. Wash 150 mm dishes of 80% confluent parental and KO lines with 10 ml of warm PBS 1x.
v. Add 5.0 ml of warm trypsin to both the parental and the KO cell dishes.
vi. Incubate dishes at 37°C, 5% CO2 for 2 min in a cell culture incubator.
vii. Confirm that cells have detached from the plate by using a bright field microscope, or visually inspect the plate. If cells are still attached, continue the incubation in the incubator until they have detached. Incubation time will vary between cell lines.
viii. Inactivate trypsin by adding 5.0 mL of complete culture media.
ix. Collect parental and KO cells in separate 15 ml canonical tubes.
x. Centrifuge at 1,500 x g for 3-5 min to pellet cells.
xi. Discard the supernatant.
xii. Resuspend the parental cell pellet with 2.0 ml of labelling culture media containing 5 µM of CellTracker Green CMFDA Dye.
xiii. Resuspend the KO cell pellet with 2.0 ml of labelling culture media containing 1 µM of CellTracker Deep Red Dye.
xiv. Incubate the cell suspensions in a cell culture incubator for 30 min with the lid slightly open. Gently tap the bottom of each tube every 5 min to put the cells back in suspension.
xv. Centrifuge both 15 ml tubes at 1,500 x g for 3-5 min.
xvi. Discard the supernatant.
xvii. Resuspend each labelled cell pellet with complete medium and count the cells.
xviii. Prepare a master mix with sufficient parental:KO cells at a ratio of 1:1 to plate cells in each well of the 96-well plate.
CRITICAL STEP For most cancer cell lines, 10,000 parental cells combined with 10,000 KO cells (20,000 cells in total) leads to 50-60% confluence in a well of a 96-well plate, which is ideal for subsequent imaging.
xix. Incubate cells in 100 µl of complete cell culture medium overnight at 37°C in the cell incubator.
Fixation in 96 well plates
xx. Add 100 µl of pre-warmed (37°C) IF fix buffer on top of the culture medium. Final concentration of PFA is 4%.
xxi. Incubate for 15 min at 37°C.
xxii. Aspirate and wash 3x with 100 µl PBS at room temperature.
xxiii. Plates can be stored at 4°C for a few days. Protect plates from light. Seal the plate using parafilm to avoid evaporation.
3B) Primary antibody staining ● Timing 24 hrs (Day 2)
i. Prepare content of each well. A total of 33 wells is required to test 14 antibodies, at two different concentrations, together with necessary controls.
CRITICAL STEP Wells #1-28 are dedicated for testing primary antibodies, whereas wells #29-33 are dedicated for controls. Wells #31 & #33 address bleed-through from the channel 1, 2 and 4 into channel 3. Well 32 controls DAPI emission bleed through into Channel 3.
ii. Incubate the necessary wells from the 96-well plate prepared in Step 1 with 100 µl of IF permeabilization buffer for 10 min at room temperature.
iii. Wash wells three times with 100 µl of 1x PBS.
iv. Incubate wells with 100 µl of IF blocking buffer for 30 min at room temperature.
v. During the incubation, prepare a 100 µl dilution of each primary antibody in IF buffer.
CRITICAL STEP After performing hundreds of immunofluorescence experiments, we found that 1.0 µg/ml is an appropriate first concentration to test as it generally provides an adequate signal falling within the detection range of a microscope. When an antibody is recommended for IF by the manufacturer, the recommended concentrated is tested, together with 1.0 µg/ml. If the recommended concentration is 1.0 µg/ml, 2.0 µg/ml is also tested. Where an antibody is not recommended for IF, 1.0 µg/ml and 2.0 µg/ml are tested. Antibody titration should be performed if signal obtained falls outside the linear range of detection.
vi. Incubate wells with the appropriate antibody dilution overnight at 4°C, or with IF buffer for control conditions.
3C) Secondary antibody labelling ● Timing 2 hrs (Day 3)
i. Wash wells three times for 10 min with 100 µl of IF buffer.
ii. Incubate wells with secondary antibody dilution containing either 0.1 µg/ml of goat secondary antibodies coupled to Alexa 555 in IF buffer, or with IF buffer alone for specific control wells.
iii. Wash wells three times for 10 min with 100 µl of IF buffer.
iv. Incubate wells with 1x PBS containing 5 ng/ml of DAPI, except specific control wells.
v. Wash twice with 100 µl of 1x PBS.
3D) Cell imaging ● Timing 2 hrs (Day 3)
i. Image wells (see raw images in Fig. 10a).
CRITICAL STEP We use an ImageXpress micro widefield high-content microscope equipped with 395, 475, 555 and 635 nm solid-state LED lights and bandpass filters to excite and capture separately DAPI, CellTracker Green CMFDA, Alexa568 (Alexa fluor 555) and CellTracker Deep Red respectively. The filter cube specifications are the following: 1) excitation (The excitation spectra are based on the emission band of the light sources (Lumencore AuraIII): blue - 395/25, green - 475/28, red - 555/28, far red - 635/22, 2) emission: blue - 432/36, green - 520/35, red - 600/37, far red - 692/40. The objective used is a water Apo LambdaS LWD with magnification of 20X, NA 0.95. The LED light or Illumination power is attenuated or optimized based on the signal expression for different targets for each channel. Camera is a 16-Bit CMOS 1.97mm FOV are used with a binning of 2, with a calibration (binned) of 0.6792 X 0.6792 um. The target maximum intensity is set at 33000. The Z-series is set at 2D projection image only. The average number of WT and KO cells imaged per condition is minimally 500.
CRITICAL STEP Sequential imaging setup is preferred to avoid any bleed through between channels. To control for bleed through when imaging 3 or 4 channels, four different controls are needed for each imaging experiment. Use the same setting for imaging the control samples. Image the single labelled control with all filters sets and carefully analyze potential bleed-through in the unlabelled channels. Adjust the emission spectra for each channel so that there is no/minimal bleed through into the unlabelled channels. To control for auto fluorescence, image an unlabelled cell sample that has gone through the staining protocol, in each of the four channels.
3E) Image analysis ● Timing 2 hrs (Day 4)
i. Image analysis can be done either visually or using the following analysis process.
CRITICAL STEP Antibody performance can be assessed visually. Antibodies that can immunolocalize their protein target will generate a specific signal in the parental cells, and a signal in KO cells comparable to the image background (area outside a cell). For a larger initiative, the automatic cell segmentation and quantification allow a more robust and reliable comparison of antibody performance between antibodies since antibody signals can be measured from thousands of cells.
ii. Using Fiji, inspect cell mask channels in images from several wells to make sure the cell mask staining is visible above background and noise, and determine an approximate cell diameter (in pixels) using the ellipse tool.
iii. To segment cells, run the cellpose segmentation36 pipeline on cell mask and DAPI images. It is highly recommended to set up cellpose in a conda environment on a CUDA-capable GPU-equipped system, and to use a script to batch process all images from all wells from a plate. Use the cell diameter estimated above (in ii) as an input parameter and choose the cytosol-specific (‘cyto’) model. The output images after running cellpose are labeled masks of cells detected in each image, and can later be used as masks for antibody staining intensity quantification (Fig. 10b). For this step, we provide a script written in python (cellpose_batch_ycharos_IMX_images.py).
iv. In the raw images, estimate the base image background in the antibody channel. This can be done with a minimum intensity projection of images of an empty well, or by generating a minimum intensity projection of several sparsely seeded wells. This can be generated using a helper script (minimum_intensity_projection_images.ijm). Calculate the median intensity of this base background.
v. The following steps (v, vi, and vii) can be performed using a Fiji script (main_ycharos_IMX_images_script_Fiji.ijm) For each antibody image, generate a thresholded binary image by first calculating a pixel intensity value threshold using the Otsu method and converting the intensity image to a binary masks image.
vi. For each antibody image calculate the median intensity for all pixels outside of cellpose and Otsu thresholded objects and divide that intensity by the base background median intensity. Multiply the base background image by that ratio and subtract that resulting image from the antibody image. The resulting image is a background-subtracted antibody staining image, based on the background image obtained from an empty well, scaled to within image background intensities.
vii. In the background-subtracted antibody images, measure the intensity and dimensions statistics (mean, sd, median, area, xy coordinates, etc) for each mask.
viii. Generate cropped images with cell mask outlines overlaid on antibody images and on DAPI images, respectively (Fig. 10c, d), using a helper script in Fiji (crop_and_make_figure_panels.ijm)
ix. With the data table, calculate the ratio mean parental intensity divided by mean KO intensity for each image, plot these ratios for each image, for each antibody tested (Fig. 10e). This can be calculated in any data analysis software, here we have provided an R script for this purpose using data tables generated in previous scripts (calculate_ratios_and_plotting_template.R).
