Assessing mRNA translation in mouse adult microglia and bone-marrow-derived macrophages

Summary Protein synthesis, or mRNA translation, is the biological process through which genetic information stored in messenger RNAs is encoded into proteins. Here, we present an optimized protocol for assessing the translation rate in mouse adult microglia and cultured bone-marrow-derived macrophages. We describe steps for isolating cells, treating them with a puromycin-analog probe, and fluorescently labeling the puromycylated-polypeptide chains. We then detail their quantification by flow cytometry or with a fluorescent plate reader. For complete details on the use and execution of this protocol, please refer to Keane et al. (2021).1

Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Ignazio Antignano, Lily Keane, Melania Capasso ignazio.antignano@dzne.de (I.A.) melania.capasso@dzne.de (M.C.) Highlights A fast and easy protocol to assess translation rate in ex vivo microglia and BMDMs Fluorescent readout measured by flow cytometry or a plate reader Flow cytometry option allows simultaneous detection of surface or intracellular markers Avoids use of radioactive tracers marrow-derived macrophage (BMDM) cultures, the growth factor M-Csf (Macrophage colony-stimulating factor, or Csf-1), which promotes monocyte differentiation into macrophages, is required.This can be obtained by supplementing the culture media with supernatant from L929 cells, which naturally produce M-Csf. 7The L929 supernatant needs to be prepared prior to starting bone marrow isolation.The protocol used for L929 supernatant production is described in ''materials and equipment''.
Finally, we have not previously validated this assay in other cell types, nonetheless, this protocol might represent a valuable starting point to assess translation rate in other cells of interest.

Timing: 3-4 h
This section describes the isolation of mouse brain cells, which will be used to assess translation in adult microglia.
1.If required, treat mice according to your experimental protocol (i.e., intraperitoneal injection of 5 mg/kg LPS for 4 h).2. Sacrifice animals according to your institutional permissions.
Optional: Mice can be perfused before brain cell isolation to remove peripheral blood cells, nonetheless, this step is not essential, since it is possible to gate on microglia in the flow cytometry analysis.

Remove brain and process it according to the Adult Brain Dissociation protocol from Miltenyi
Biotec (https://www.miltenyibiotec.com/DE-en/products/adult-brain-dissociation-kit-mouse-andrat.html#130-107-677).Troubleshooting 4 and troubleshooting 5. Ensure to remove debris using the debris removal step according to manufacturer's guidelines but skip the Red Blood Cell Lysis step.
Note: we do not perform the Red Blood Cell Lysis step, since in our experience, this can reduce yield and viability of our cells of interest.If mice are not perfused, red blood cells remain in the cell suspension; they do not interfere with the experimental protocol and are then gated out in the flow cytometry analysis.
Note: Cells isolated from only one of the two brain hemispheres will be sufficient for the assay.
4. Count cells using an automated cell counter or a hemocytometer. 5. Adjust brain cell suspension to 2 3 10 6 cells/100 mL in microglia medium (MM).6. Proceed to determine translation rate as described below in the flow cytometry-based translation assay in adult microglia paragraph -step 1.

Timing: 7 days
The following describes the isolation and cell differentiation of bone marrow-derived macrophages (BMDMs) generated according to the protocol by Joachim Weischenfeldt and Bo Porse, 8 with minor changes, as described below.The L929-derived conditioned medium used for bone marrow cell differentiation into macrophages is obtained by harvesting the supernatant of L929 cells, which secrete the growth factor M-Csf 7 and should be prepared in advance as described in ''materials and equipment''.

MATERIALS AND EQUIPMENT
Materials relative to the Protein Synthesis Assay described below can also be found in the corresponding assay protocol (https://www.caymanchem.com/product/601100/protein-synthesis-assaykit).Since we have slightly modified the manufacturer's protocol, in order to evaluate the translation rate of adult microglia cells from multiple samples using a V-bottom plate instead of single tubes, we report the preparation of materials specific to our assay.
The equipment needed for the execution of this assay are described in the section ''Others'' in the key resources table.

Microglia medium (MM)
Prepare a solution containing DPBS (with calcium, magnesium, glucose, and pyruvate) and 0.5% fetal bovine serum (FBS).Store it up to 2 months at 4 C and pre-warmed in a water bath at 37 C prior to using it.Thaw the solution when needed in a water bath at 37 C. Use within maximum one day after thawing.

L929-conditioned medium (L929CM), only for BMDMs
The L929-conditioned medium can be generated according to the protocol by Joachim Weischenfeldt and Bo Porse, 8 with minor changes, described below.
Seed 0.47 3 10 6 L929 cells in a 75 cm 2 flask containing L929M (final volume 55 mL) and grow cells for 7 days in a 37 C/5% CO 2 incubator.At day 7, collect supernatant, filter with a 0.2 mm pore filter and prepare aliquots for freezing.
Store frozen aliquots at -80 C. When needed, thaw aliquot in a water bath at 37 C and use within 1 day.

Dissociation buffer (DB), only for BMDMs
Dilute Cell Dissociation Buffer in pre-warmed DPBS (without calcium, magnesium) at 1:1 ratio.Prepare enough dissociation buffer for the total amount of plates to be harvested.Once diluted for the amount needed, Cell Dissociation Buffer has to be used within one day.

Assay Buffer (1X)
Dilute the Cell-Based Assay TBS (10X) contained in the assay kit with 90 mL of cell culture water.Mix well at 18 C-24 C. The diluted Assay Buffer should be stable for at least one year when stored at 18 C-24 C.

Cycloheximide solution, only for adult microglia
Prepare a solution containing 100 mg Cycloheximide in 100 mL of microglia medium (MM) by diluting 1:500 a stock solution of 50 mg/mL for each sample.Prepare enough solution based on the number of samples to be pre-treated with Cycloheximide.
Vortex and keep it at 18 C-24 C.
Note: Cycloheximide is dissolved in DMSO and stored at -20 C. Keep the vial at 18 C-24 C for at least 30 min and vortex prior to using it.
O-Propargyl-Puromycin (OPP) stock solution, only for adult microglia The following amount refers to a total well volume of 110 mL per sample, which includes the 10 mL of OPP stock solution that needs to be added to each of the indicated conditions.Multiply the following reagents described in the table by the total number of conditions and samples according to the experimental design.The following amount refers to a total well volume of 120 mL per one sample, which includes the 10 mL of FACS stock solution that needs to be added to each of them.Multiply the following reagents described in the table by the total number of samples/conditions according to the experimental design.
Note: We incubate FcR block (TruStain FcX) together with primary antibodies against CD11b and CD45.This strategy, designed to reduce incubation times, works with these antibodies but should be tested if users utilize different ones.

Cell-based assay 5 FAM-Azide Staining Solution
The following amount refers to a single condition for one sample.Multiply the following reagents described in the table by the total number of conditions and samples according to the experimental design.Protocol STEP-BY-STEP METHOD DETAILS Each sample will require the following conditions: 1) cells treated with Cycloheximide (CHX) and OPP (hereafter defined as ''CHX+OPP'') as a control for reduced translation; 2) cells treated with OPP (hereafter defined as ''OPP''); 3) cells not treated with OPP but subsequently treated with FAM-Azide (defined as ''no OPP''); 4) cells used as ''unstained control'' and ''live/dead control'', only for FACS analysis (Figure 1).
Note: If the number of cells is limited, conditions 3) and 4) can be obtained by mixing cells from multiple samples.

Flow cytometry-based translation assay in adult microglia
Timing: 5 h The following protocol describes the steps needed for determining the translation rate in adult mouse brain cells.Previous steps 1-6 were described in section ''before you begin'', isolation of brain cells from adult mice.
1. Plate 100 mL of brain cell suspension per condition in a V-bottom plate.

Reagent Dilution factor Amount
Cell-Based Assay Buffer (1X) -98.8 mL Total N/A 100 mL Prepare the solution immediately before use, protected from light and store at 18 C-24 C. Mix well and avoid generating bubbles when pipetting up and down with a P200 pipette.b.Add 100 mL of Microglia medium (MM) to all other samples and resuspend with a P200 pipette.c.Incubate cells in a cell culture incubator at 37 C for 15 min.After treatment, proceed immediately to point 4.

O-Propargyl-Puromycin (OPP) incubation
a. Add 10 mL of OPP stock solution only to samples designated as ''OPP'' and ''CHX+OPP''.Mix well by pipetting up and down without generating bubbles.b.To the remaining samples, add 10 mL of media.Incubate cells in a cell culture incubator at 37 C for 1 h.Each sample now has a final volume of 110 mL.
Note: the FACS staining stock solution can be prepared 10-15 min before the end of the OPP treatment.
5. Surface marker staining.a. Add 10 mL of the previously prepared FACS staining stock solution to each condition (no OPP, OPP and OPP + CHX) but not the unstained controls.b.Mix to get a single cell suspension by gently pipetting without generating bubbles.c.For the unstained control well, use 10 mL of MM only and for the live/dead control, use 10 mL of MM containing only the live/dead staining dye.d.Protect plate from light and incubate for 10 min at 37 C. e. Spin down the plate at 400 3 g for 5 min at RT and carefully aspirate the supernatant.
Note: If channel compensation on the flow cytometer is not performed with beads, samples with single stains and Fluorescent Minus One (FMO) stains will be required.
CRITICAL: Fluorescently labeled antibodies used for FACS analysis are light-sensitive.Therefore, it is advisable to perform the following steps shielding samples from light.In order to achieve this, cover the plate with parafilm (to avoid spillages in neighboring wells) or a 96-well plate lid; then cover the plate with aluminum foil.CRITICAL: Aspirating supernatant with a vacuum pump should be avoided in order to reduce the risk of aspirating cells.This step can be carried out with a pipette, using a multichannel pipette if working with a considerable number of samples.The same applies to the subsequent steps.Note: Cell-Based Assay Wash Buffer contains 0.1% of Polysorbate 20 (also known as Tween 20), a nonionic surfactant required for cell permeabilization, 9 in order to allow FAM-Azide to enter cells and encounter OPP-conjugated nascent peptides.Optional: To maximize cell recovery in step 8f, resuspend cells in 100 mL of Assay Buffer (1X); transfer sample to the corresponding FACS tube and keep the tube on ice and in the dark.
Then use an extra 100 mL of Assay Buffer to further wash the well, collecting any remaining cell and transferring it into the corresponding sample tube.
In-vitro translation assay for cultured bone marrow-derived macrophages (BMDMs)

Timing: > 5 h
This section describes the steps needed for determining the translation rate in differentiated BMDMs.Previous steps 1-26 were described in section ''before you begin'', bone marrow-derived macrophages culture protocol.
1. Spin down the plate with BMDMs at 400 3 g for 5 min at 18 C-24 C, carefully aspirate the supernatant and proceed immediately to the next step.Troubleshooting 3.
Note: Although BMDMs are strongly adherent cells, we follow the Protein Synthesis Assay manufacturer's recommendation to centrifuge the plate, as described in the assay protocol related to plate reader detection.
Note: if working with a considerable number of samples, the supernatant can be removed more quickly by using a multichannel pipette.The same applies to the subsequent steps.
Note: For BMDMs, no pre-treatment with Cycloheximide with our time point of 3 h was required.a. Add 100 mL of Cell-Based Assay Wash Buffer provided with the kit and incubate the cells at 18 C-24 C for 5 min.b.Spin down the plate at 400 3 g for 5 min at 18 C-24 C and carefully aspirate the supernatant.c.Repeat washing steps (4a and 4b) for a total of three times.
Note: Cell-Based Assay Wash Buffer contains 0.1% of Polysorbate 20 (also known as Tween 20), a nonionic surfactant required for cell permeabilization, 9 in order to allow FAM-Azide to enter cells and encounter OPP-conjugated nascent peptides.
Note: During the last centrifugation steps (4a-c), start preparing the 5 FAM-Azide staining solution in a tube protected by light.
CRITICAL: FAM-Azide is light sensitive.All the next steps must be performed without direct exposure to light.Perform incubations in the dark.

FAM-Azide staining.
a. Add 100 mL of 5 FAM-Azide Staining Solution and incubate cells for 30 min in the dark at 18 C-24 C. b.Spin down the plate at 400 3 g for 5 min at 18 C-24 C and carefully aspirate the supernatant.c.Add 100 mL of Cell-Based Assay Wash Buffer provided with the kit and incubate the plate protected from light at 18 C-24 C for 5 min.d.Spin down the plate at 400 3 g for 5 min at 18 C-24 C and carefully aspirate the supernatant.e. Repeat cell washes steps (5c and 5d) for a total of three times.f.Add 100 mL of previously prepared Assay Buffer (13).Proceed immediately to measure the fluorescence intensity with a fluorescent plate reader using a filter designed to detect FITC (excitation/emission = 485/535 nm).Troubleshooting 2.

EXPECTED OUTCOMES
The expected results of both assays are shown below (Figure 2).In Figure 2A, microglia are selected according to the following gating strategy: a) in a side scatter (SSC-A) -forward scatter (FSC-A) plot, brain cells are selected in order to exclude debris, dead cells and red blood cells; b) a FSC-H -FSC-A scatter plot allows the identification of single cells vs. doublets or multiple cells adhering to each other; c) signal from LIVE/DEAD Fixable Near-IR viability dye allows to identify dead cells that take up the dye vs. healthy cells that do not; d) microglia are identified as CD11b high and CD45 int cells; e) translation rate is estimated through the detection of 5-Carboxyfluorescein (5-FAM) at the excitation wavelength of 485 nm and emission wavelength of 535 nm.
In Figure 2B, the plate reader relative fluorescent intensity (RFU) gives an estimate of translation rate in BMDMs.
In both cases, Cycloheximide treatment confirms that the detected fluorescence diminishes in the presence of a translational inhibitor.The extent of inhibition with CHX treatment is different between BMDMs and microglia and this might be due to different factors, such as incubation time with CHX, which is shorter for microglia.When designing the protocol for microglia, we aimed to reduce incubation times as much as possible, in order to reduce any potential loss of viability while microglia are in suspension.We did not aim to completely inhibit protein synthesis with our settings; it is possible that a higher concentration of CHX, in addition to longer incubation times, would completely inhibit protein synthesis, however, we have not tested them out of concern for microglia viability.

QUANTIFICATION AND STATISTICAL ANALYSIS
Flow cytometry data analysis of translation rate in adult microglia can be carried out with FlowJo software (v10, BD Biosciences).A general guideline of data analysis is described below: 1. Run FlowJo (or alternative flow cytometry analysis software) on a computer.2. Import .fcsfiles exported from the flow cytometer in FlowJo.
3. Apply compensation if needed.4. Gate cells according to the gating strategy described in Figure 2A and identify microglia based on the expression of the CD11b and CD45 markers. 5. Determine the median fluorescent intensity (MFI) of 5-FAM signal within the CD11b high CD45 int microglia population as a readout for translation rate.
Fluorescence intensity of protein synthesis in BMDMs can be easily determined by subtracting the blank (media only) from each condition in an excel file exported from a fluorescent plate reader.Statistical analysis and data visualization can be performed using GraphPad Prism.

LIMITATIONS
The translation rate refers to the speed at which ribosomes translate the genetic information contained in mRNA molecules into proteins.This process can vary based on the cellular context and cell type being investigated.It is possible that the translation rate of microglia and BMDMs from certain genetic and/or disease models is too low to be detected and might be indistinguishable from the no OPP negative control.In this case, a possible solution might be to stimulate cells with growth factors that activate pathways promoting protein synthesis, such as the insulin growth factor-1 (IGF-1) for mTORC1-dependent translation.
Furthermore, in case it is not possible to use an adult brain cell suspension, neonatal primary microglia cultures could be used as an alternative, however, we have not previously tested it and some optimization might be required.

TROUBLESHOOTING Problem 1
Cell-based assay fixative provided by the assay kit might lead to inconsistent results (flow cytometrybased translation assay in adult microglia -step 6; in-vitro translation assay for cultured bone marrow-derived macrophages (BMDMs) -step 3).

Potential solution
We have encountered problems when using the assay fixative provided with the protein synthesis assay kit.This might be related to the formaldehyde solution not being stable when not freshly prepared.We solved this issue by replacing the assay fixative with a freshly prepared solution of 4% PFA in PBS (pH 7.4) that we routinely use for immunostaining assay.

Figure 1 .Protocol 2 .
Figure 1.Visual summary of steps required in the protein synthesis assay Image created with BioRender.com.

6 .
Cell Fixation.a. Resuspend cells in 100 mL of 4% PFA/PBS per well.Mix gently to ensure a single cell suspension.Protect plate from light and incubate at 18 C-24 C for 5 min.Troubleshooting 1. b. Spin down the plate at 400 3 g for 5 min at 18 C-24 C and carefully aspirate the supernatant.7. Washing cells with Cell-Based Assay Wash Buffer.a. Resuspend cells in 200 mL of Cell-Based Assay Wash Buffer provided with the kit.Mix to ensure a single cell suspension and incubate the plate protected by light at RT for 5 min.b.Spin down the plate at 400 3 g for 5 min at 18 C-24 C and carefully aspirate the supernatant.c.Repeat cell washes steps (7a and 7b) for a total of three times.

8 .
FAM-Azide staining.Note: During the last centrifugation steps (7a-c), start preparing the 5 FAM-Azide staining solution in a tube, protected from light.CRITICAL: FAM-Azide is light sensitive.Carry out the following steps without direct exposure to light.a. Gently resuspend cells in 100 mL of 5 FAM-Azide Staining Solution in order to obtain a single cell suspension.Incubate cells for 30 min in the dark at 18 C-24 C. b.Spin down the plate at 400 3 g for 5 min at 18 C-24 C and carefully aspirate the supernatant.c.Resuspend cells in 200 mL of Cell-Based Assay Wash Buffer provided with the kit.Mix gently to ensure a single cell suspension and incubate the plate protected from light at 18 C-24 C for 5 min.d.Spin down the plate at 400 3 g for 5 min at 18 C-24 C and carefully aspirate the supernatant.e. Repeat washing steps (8c and 8d) for a total of three times.f.Resuspend cells in 200 mL of previously prepared Assay Buffer (1X).Mix well by pipetting up and down to get a single cell suspension.Transfer the sample to a FACS tube and proceed immediately to analyze the sample by Flow cytometry.An example of the gating strategy is reported in Figure 2. Troubleshooting 2.

Figure 2 .
Figure 2. Expected outcomes of the protein synthesis assay in untreated (no OPP), Cycloheximide-and OPP-treated (CHX+OPP), OPP-treated (OPP) adult microglia or BMDMs (A) Gating strategy applied for the identification of adult microglia cells: a) Forward scatter (FSC-A) -Side scatter (SSC-A) plot of cells contained in brain cell suspension; b) FSC-H -FSC-A plot to identify single cells; c) SSC-A -LIVE/DEAD plot to identify viable cells (=negative cells) ; d) dot plot for CD11b and CD45 channels, microglia are identified based on their high expression of CD11b and intermediate expression of CD45; e) histograms for 5-FAM channel; top panel: cells with no OPP and therefore no staining; middle panel: cells with OPP but treated with CHX, showing reduced translation when compared with cells not treated with CHX (bottom panel); bottom panel: cells treated with OPP showing full translation rate.Median Fluorescence Intensities (MFI) of no OPP, CHX+OPP and OPP are reported in in the table below the histograms.(B) Relative fluorescence intensity (RFU) of the protein synthesis rate in wild-type BMDMs at steady state and after stimulation with 100 ng/mL LPS for 3 h and assessed with a fluorescent plate reader.Data adjusted from Keane et al. 1 and displayed as mean G SEM (n = 3).P adjusted value (P): **P < 0.01, ****P < 0.0001, 2-way ANOVA with Tukey correction for multiple comparisons.

TABLE REAGENT
Prepare the solution in advance to rapidly add it to each well.The solution can be stored at 18 C-24 C and needs to be used immediately.Mix the solution avoiding generating air bubbles.Prepare the solution in advance and store at 4 C up to 2 months.FACS Buffer must be ice-cold during the preparation of the FACS staining stock solution.Prepare the solution immediately before use, protect it from light and keep on ice.
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