FAST FIN PROTOCOL. Fluorescence activated sorting of fixed nuclei

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1 FAST FIN PROTOCOL Fluorescence activated sorting of fixed nuclei This step by step protocol has been written to be accessible to scientists who are not necessarily experienced with flow cytometry. It is complementary to the publication Marion Poll & al, EJN This protocol takes 2 consecutive working days. REQUIRED MATERIAL Reagents Iodixanol solution, 60% weight/vol (Optiprep, light sensitive) Anaesthesia (pentobarbital) Antibodies and their control IgG (see Antibody section for help with the choice) Sucrose KCl NH 4 Cl Tris HCl ph 7.4 MgCl 2 (choose hexahydrate as anhydrous will hydrate over time) Bovine serum albumin (IgG free is preferable) DNA dye (for example Hoechst 33258) Triton X 100 Paraformaldehyde (in order to make 4% (weight/vol) formaldehyde solution) PBS Equipment Access to a flow cytometry analyzer or sorter depending on the application Refrigerated centrifuge with a swinging bucket rotor to be run at 4 C 10,000 x g (clearing factor k = 1600) with corresponding transparent tubes (translucent tubes will not fit) Note: The swinging bucket centrifuge is preferable but not compulsory (see alternative protocol option #2) Table top refrigerated centrifuge for 1.5 ml tubes, to be run at 4 C 10,000 x g Rotating wheel in a cold room at 4 C for tube incubation Pump for perfusion Dissection tools 2 ml Dounce homogenizer with pestle A (clearance µm) and pestle B (clearance µm) Low binding pipet tips (with filter), they are preferable to reduce nuclei loss Syringes (to anaesthetize the animals) 1

2 SOLUTIONS HOMOGENIZATION 50 mm sucrose 25 mm KCl 5 mm MgCl 2 50 mm NH 4 Cl 120 mm Tris ph 7.4 WORKING SOLUTION 150 mm KCl 30 mm MgCl mm Tris ph 7.4 DILUENT 250 mm sucrose 25 mm KCl 5 mm MgCl 2 20 mm Tris ph 7.4 RESUSPENSION Diluent solution + 1% (wt/vol) BSA DNA LABELING Resuspension solution µg/ml Hoechst (or another DNA dye, depending on your choice of fluorophores and lasers/filters available on your flow cytometer) PERMEABILIZATION Resuspension solution + 0.1% (vol/vol) Triton X 100 Notes: All solutions should be sterile filtered and kept at 4 C at all times to avoid contaminations. Sucrose containing solutions should be discarded 1 week after the bottle has been opened. All solutions containing iodixanol, DNA dye or fluorescent antibodies should be protected from light. When studying phosphorylation, phosphatase inhibitors should be added (for example 2 mm imidazole, 1 mm sodium fluoride, 1.15 mm sodium molybdate, 1 mm sodium orthovanadate, 4 mm sodium tartrate). Depending on your study you might want to add different inhibitors. 2

3 To be prepared one day before FORMALDEHYDE SOLUTION 4% (weight/vol) formaldehyde PBS 1X final ph 7.4 (can be prepared one day in advance, stored at 4 C, to be used at room temperature) 50% IODIXANOL SOLUTION Mix 1 volume of Working solution with 5 volumes of Optiprep (iodixanol 60%) 43% IODIXANOL SOLUTION Mix 86% (vol/vol) of the 50% iodixanol solution with 14% of the Diluent solution (for 5 ml mix 4.3 ml of 50% iodixanol solution with 700 µl of Diluent). 22% IODIXANOL SOLUTION Mix 44% (vol/vol) of the 50% iodixanol solution with 56% of the Diluent solution (for 20 ml mix 8.8 ml of 50% iodixanol with 11.2 ml of Diluent) Note: Make sure to fully homogenize solutions containing iodixanol before use. Protect from light. 3

4 FAST FIN PROTOCOL DAY 1 Before the tissue preparation, you might perform the in vivo protocol of your choice with the animals (injections, behavior, ). Note: If you are using transgenic animals expressing fluorescent proteins, you will need a control animal which does not express any fluorescent protein in order to identify your positive population by comparison. Set the centrifuges at 4 C. FORMALDEHYDE FIXATION The aim of this step is to have a sufficient but not excessive fixation, in order to dissociate the tissue without destroying the nuclei while avoiding protein leakage. The following fixation protocol is optimized for adult mice. If you wish to use juveniles or different animal species, we recommend you use the standard flow rate in your case (as if for immunohistochemistry). Concerning the fixation duration, it should be in the same range, but if you notice that the tissue is too hard to dissociate as compared to ~ 25 mm 3 of adult mouse brain (whole hippocampus or whole striatum), you need to reduce the fixation duration (to be able to dissociate the tissue, without destroying the nuclei). If it is too easy you should increase fixation time (to avoid protein leakage). You might need to dissect a region of interest bigger than ~ 25 mm 3. In that case we recommend splitting the tissue into smaller pieces for homogenization in several Dounce homogenizers or using a larger Dounce homogenizer (same clearance of the pestles). Let the formaldehyde solution equilibrate to room temperature. For this step you will need: dissection tools, a pump, some needles, a timer, an ice bucket, Dounce homogenizers with pestles A and B, 1 ml of homogenization solution per sample (4 C), 1.5 ml Eppendorf tubes (one per sample), anesthetic, syringes Fill the tubing of the pump with formaldehyde, make sure there are no bubbles and set the pump to a flow rate of 20 ml/min (you might need to measure and adjust flow rate empirically). Fill one Dounce homogenizer with 1 ml of homogenization solution and have the corresponding labeled Eppendorf tube ready, on ice. Deeply anesthetize the first animal (500 mg/kg pentobarbital, i.p.). Once its reflexes are abolished (eyes, tail, feet), start the transcardiac perfusion immediately with formaldehyde (if you re studying phosphorylation, this is important to preserve it). Start the timer. Make sure the incision of the right atrium of the heart is sufficient, to avoid overpressure. After 5 min, stop the pump (but not the timer). At this point, depending on the time required for dissection, you might need to wait a little then dissect your tissue of interest, so that you 4

5 dip it into the homogenization solution in the Dounce homogenizer at exactly 9 min (precise timing is important to avoid excess drying of the sample). Make ~ 25 strokes with pestle A. Do not press too much on the pestle; damage would lead to loss of some nuclei. The correct pressure is the lowest necessary to allow correct dissociation of the tissue. Avoid making bubbles, keep the end of the pestle in the solution. Stop using pestle A when strokes no longer improve dissociation. At this point you still have a lot of small but visible pieces of tissue. Make ~ 25 strokes with pestle B. Same advice. At the end you should not have any visible piece of tissue but a cloudy solution. Note: If the homogenization is not sufficient, you will have a lot of aggregates and risk to block the tubing of the flow cytometer. If homogenization is excessive, it will result in poor yield and can bias the proportion of cell types (the cells with bigger nuclei will be damaged and lost more easily). Transfer the homogenate into an Eppendorf tube and leave it at 4 C. Protect from light if using fluorescent proteins. Proceed with the other animals. Rinse the Dounce homogenizers and the pestles with distilled water as soon as you are finished. It is important that they stay very clean so that the clearance between pestle and cylinder is preserved. You can wash them later with detergent and bleach and rinse thoroughly. NUCLEI PURIFICATION Two options are presented. The first one leads to the purest preparation, whereas the second one does not require the use of a centrifuge with a swinging bucket rotor. Centrifuge the samples at 2,000 x g 5 min 4 C Discard the supernatant completely and resuspend the pellet in 1 ml of the 22% iodixanol solution (for ~ 2 million nuclei and 4 ml centrifuge tubes, otherwise scale accordingly). Pipet up and down 15 times (pipet everything from the bottom, tilt your pipet facing the wall of the tube without touching it and eject the solution without making bubbles always proceed this way when resuspending nuclei). If the solution is not well resuspended you can lose a lot of nuclei at this stage when they are clustered with membrane debris. On the other hand you should always manipulate the nuclei as gently as possible because they are fragile. Never add solution directly targeting the pellet. Regular protocol, option #1: Layer 500 µl of 43% iodixanol at the bottom of the 4 ml centrifuge tube (this volume can be adjusted if you use bigger or smaller centrifuge tubes). You can draw a line on the tube to delineate the upper surface of this phase as it will be almost invisible afterwards. Add some 22% iodixanol solution very slowly and carefully (to avoid a mix of the two phases). Let the solution flow slowly on the wall of the tube. Keep the tube straight. Try not to disturb 5

6 the 43% iodixanol phase. The less they mix the better the separation will be and the easiest it will be to recover the nuclei. Then add the homogenate slowly. Fill the centrifuge tube completely (add 22% iodixanol solution). Equilibrate the weight of the tubes and their swinging buckets which will face each other, before running the centrifuge. Add 22% iodixanol solution if necessary. Centrifuge 30 min 10,000 g 4 C (adjust time if using a different rotor, k = 1600). Remove the upper fatty layer: quickly dip into it a pipet tip by hand, it should stick to this and hence be efficiently discarded. Discard most of the 22% iodixanol layer and collect the interface between 22% and 43% iodixanol (we usually collect 500 µl). This is where the nuclei should be present. Put them into a new tube. Dilute the collected nuclei fraction with resuspension solution. The dilution factor should be at least 1:2 but 1:3 will be more efficient to pellet the nuclei. Invert the tubes 7 times to ensure a proper mixture (important for correct nuclear pelleting). Centrifuge 10 min 10,000 g 4 C. At this stage you might see a very light pellet (it depends on the quantity of nuclei). It is also possible that no pellet is visible even if you have pelleted nuclei. If the dilution was not sufficient or if the homogenization was incomplete the nuclei will not pellet properly and you can lose them at this step. Discard supernatant; be very careful to leave the pellet (you can leave a drop of supernatant if you are not sure). Alternative protocol, no rotor with swinging buckets, option #2: Note: This option will lead to a preparation which is less pure but you can have a better yield because you pellet all the nuclei instead of having them at the interface where they are more difficult to collect. You should absolutely avoid having unwanted solid contaminants in your homogenate because with this alternative they will not be removed. The risk of blocking the tubing of the flow cytometer is higher in this case and you should consider filtering your samples before injecting them into the machine. Centrifuge the Eppendorf tubes for 10 min 10,000 g 4 C. Remove the fatty upper layer with a pipet tip by hand (see option 1 for details). It is very likely that you cannot remove everything at once and some of it will stick to the wall of the Eppendorf. Detach the residual material from the wall of the tube. 6

7 Centrifuge again 10 min 10,000 x g 4 C and remove again the upper layer. Repeat this a third time. At this point you should have removed entirely the upper layer. If not, you can add a 5 minute centrifugation step for all samples. The nuclei should form a pellet, visible or not depending on the quantity. Remove the supernatant. DNA LABELING Resuspend the nuclei in the DNA labeling solution by pipetting up and down 15 times (same method as before). We recommend 1 ml for ~ 2 million nuclei (25 mm 3 of starting tissue). Scale accordingly. During the 20 min incubation, put the samples on a slow rotating wheel at 4 C. Centrifuge 5 min 2,000 g 4 C. Discard supernatant (you can leave a little bit of it to be safer with the nuclei but always be very consistent among samples). If you do not need to immunolabel, add some new resuspension solution (1 ml) and invert the tubes. Centrifuge again 2,000 g 5 min 4 C, discard supernatant, resuspend in resuspension solution by pipetting up and down 15 times. Your samples are now ready for flow cytometry. Follow the corresponding instructions. You can proceed the same day (better) or the following day (keep samples at 4 C, resuspend the nuclei 1 h before flow cytometry) If you want to proceed with immunolabeling, directly keep on with the corresponding instructions. ANTIBODY LABELING Choice of antibodies: Antibodies which have already been validated for flow cytometry or immunofluorescence on fixed tissue are the most likely to work in this protocol. Primary antibody already conjugated with fluorophore or primary + fluorescent secondary? Using an already conjugated primary antibody is very common in flow cytometry. Labeling will be quicker (and you can lose less nuclei because there are less washing steps) but it will be difficult or impossible to have a correct control antibody (i.e. non specific antibody of the same isotype, at the same concentration, conjugated with the same fluorophore with the same molar ratio antibodyfluorophore). In this case you must have negative control nuclei to check for non specific binding. Primary + fluorescent secondary will require more steps. It will be easier to have a good control with an unconjugated primary antibody of the same isotype (with no specificity) + the same fluorescent secondary. 7

8 Fluorophores You should choose your fluorophores in accordance with the parameters of your flow cytometer. If you perform multiple labeling, check for spectra overlap, you might need to use signal compensation. The personnel of the flow cytometry facility can give you advice for your particular study. Controls If you have negative control cells you should use them to check for non specific labeling of your antibody. If you are using a non conjugated primary + fluorescent secondary antibody, you should have a control primary antibody of the same species and isotype to be used at the same concentration (weight/vol) with the fluorescent secondary. For each nuclear preparation, keep a sample to process through all the steps but without antibodies. It will be useful to set the parameters of the flow cytometer (except those which are specific for the fluorophores) without wasting precious labeled samples. Moreover it is useful to quantify the nonspecific binding of the primary control IgG + secondary. For each nuclear preparation the background fluorescence can slightly vary; that s why it is better to have the information for all the preparations separately. Compensation Compensation refers to procedures that take into account spectra overlaps. If needed, prepare control samples labeled with only one fluorophore (+ DNA dye). For each fluorophore, take a sample which has the highest signal expected on this channel. Avoid compensations between DNA dye and other fluorophores. What is the optimal antibody concentration? Prior to your study, you should test several dilutions of each antibody (primary/control IgG, secondary). You can test series of two fold dilutions for the primary. Choose the dilution with the highest specific signal with the best signal to noise ratio. The concentration of the nuclei during optimization should be similar to the concentration you will use for your study. Resuspend samples in 1 ml of permeabilization solution. Be very gentle while pipetting because the nuclei can easily be destroyed with detergent. Incubate for 5 minutes at 4 C and centrifuge 5 min 2,000 g 4 C. Remove supernatant. Add 1 ml of resuspension solution. Note: The nuclei can be destroyed if they stand for too long in a solution containing Triton X 100 (avoid more than 10 min total before adding the new resuspension solution). Always be consistent with the permeabilization duration. After this treatment, the nuclei will be more fragile. Resuspend the samples by carefully pipetting up and down 15 times. The solution should look completely homogenous as you will then aliquot each sample into different tubes to make different labeling (at least one antibody and its control IgG). Each aliquot should contain at least 5000 nuclei from the population of interest at the end of the procedure. Note: For the study described in the paper we would make aliquots of 100 µl. 8

9 In each aliquot, add 1 volume of the primary antibody concentrated 2X in resuspension solution (final concentration 1X). Let them incubate overnight on a running wheel at 4 C. DAY 2 Proceed with two washes: centrifuge 2,000 g 5 min 4 C, discard supernatant, add resuspension solution, wait 10 minutes. Repeat one time. Note: you will not see any pellet except if you have a lot of nuclei. Depending on the tubes you use, the pellet might not stick very well to the bottom of the tube. Be very careful. You can leave some solution at the bottom (always the same quantity be very consistent between samples) and you should wash with the biggest volume possible depending on the size of your tube. Centrifuge again 2,000 x g for 5 min at 4 C. Discard supernatant. Add the secondary antibody (if necessary), in the resuspension solution. Resuspend by pipetting up and down 15 times. Incubate for 1 h 4 C on a rotating wheel. Proceed with two washes (same as before). Resuspend in resuspension solution. The final volume is critical for the subsequent flow cytometry analysis. If it is too diluted, it will take a long time to analyze all the volume. If it is too concentrated it can be diluted to fit with the flow cytometer optimal working concentration. Note: We generally use 1 ml for ~ 2 million nuclei (e.g. both striata from one mouse) FLOW CYTOMETRY Your samples should not contain any aggregate or visible debris and should be properly resuspended. We do not usually filter the samples prior to running them on the machine and never had clogging problems. However this is a serious issue concerning flow cytometry and you might want to consider filtering if you are not sure about your preparations. Some flow cytometry facilities will require you to filter anyway. Note: If you are new to flow cytometry, ask assistance from the technicians of the facility, they can do all the settings and acquisitions as long as you explain what populations you expect. Bring your samples on ice (protected from light), some resuspension solution, a pipet + pipet tips, collecting tubes (if sorting). Resuspend again each sample right before running it on the machine. Use a sample which is only DNA labeled. Adjust the voltage of the photomultiplier (PMT) of the DNA labeling channel, you should see a major population of singlets labeled and then doublets with twice the intensity (see Figure 2B in Marion Poll & al, EJN 2014). The debris which do not contain DNA have an intensity close to zero. Check the flow rate and the number of events/sec, adjust the parameters (flow rate, dilution of the samples). Select the singlet nuclei population by gating and adjust the PMTs of the side scatter and forward scatter. 9

10 For each channel you use, take the sample you consider should have the highest signal and adjust the PMT. Of course you should use only a small part of the sample and keep the rest for acquisition or sorting. Now you are ready for acquisition. You should not modify the parameters from now on. You can save them for quicker settings next time. Sorting As the diameter of the nuclei is close to 10 µm, we used a 70 µm nozzle. All collection tubes should contain a small amount of resuspension solution ( ~ 100 µl). 10

11 TROUBLESHOOTING Problem Possible cause Solution No nuclei at the end No nuclei in some samples DNA labeling intensity is variable among samples Cannot see a narrow Hoechst positive peak There is a double Hoechst positive peak (two peaks very close to each other) with similar quantities of nuclei Cannot detect GFP from GFP transgenic animal Nuclei handling is tricky because they are not visible. With time nuclei settled at the bottom of the tube and were not aspirated through the flow cytometer tubing. The nuclei concentration varies across samples and/or the volumes used for washing or the volumes left when discarding supernatant were not consistent among samples. Nuclei may be damaged and you may have fragments of nuclei. Some nuclei accumulated at the bottom of the tube since last resuspension, others were still in suspension. For this reason the DNA labeling is not homogeneous. The GFP expression is low or expressed in a very sparse population. Train with visible nuclei. After homogenization, stain the tissue with cresyl violet (or similar dye). Proceed with all the steps except flow cytometry. You should solve your problem. Resuspend with resuspension solution. If you still do not obtain nuclei it means you have lost them. If the intensity varies too much, you can make a mark on the tubes to leave always the same volume and be more consistent. If it varies a little bit it is not a problem, you can just adjust the gate of singlet nuclei for each sample. Check permeabilization step and handle the nuclei with care. Do not make bubbles, do not directly release solution on the nuclei with your pipet (but on the wall of the tube). Resuspend and then wait 15 minutes. The DNA labeling should become homogeneous. Always compare with a WT animal, the population of interest might be a very small peak as compared to the major population. If sorting, you can first select nuclei with high intensity in the GFP channel (even if no peak is identifiable). In this new fraction, the percentage of positive nuclei will be higher so the positive peak will be more visible. Note: During the second sorting the shape of the nuclei will change (they shrink in PBS) and the DNA intensity will decrease (due to dilution) but the GFP intensity will not decrease greatly. Use anti GFP antibody conjugated with FITC. 11