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CCHMC SOP Adult Mouse Type II Cell Isolation and Depletion

 

Dispase Lung Digestion

 

  1. Thaw the required volume of dispase on ice. For each mouse, add 1ml of dispase to a 14 mL round bottom polypropylene tube and place tubes into a 37oC heat block. Place the dispase needed for instilling into the lungs into an additional tube at 37o
  2. For each mouse prepare a C-tube by adding 5mL of DMEM containing 25 mM HEPES buffer and Penn/Strep.
  3. Anesthetize the mouse with 0.1mL of sodium pentobarbital. Secure animal for dissection. Spray mouse lightly with ethanol,
  4. Open the abdomen and move the organs to one side to expose the kidneys. Severe the renal artery and descending aorta to exsanguinate. Immediately raise the head of the animal. Using a strip of absorbent paper, cut to a point at both ends, remove as much blood as possible from the abdominal cavity.
  5. Open the skin from the abdomen to the top of the throat. Dissect away the salivary glands. Cut along the length of the sternum. Continue cutting to open the muscle tissue above the trachea. Remove the rib cage. Remove any muscle that obscures the trachea.
  6. Use curved forceps to pull a piece of size 2.0 suture under the trachea. Draw the ends into a loose single knot.
  7. Use a 20G, 0.75 inch angiocath catheter to cannulate the trachea. Remove the protective caps, so that just the needle and soft sheath remain. Insert the catheter into the trachea just above the cartilaginous ring, using the needle to penetrate. Stop when the needle is still visible at the bottom of the trachea.
  8. Support the catheter with one hand and carefully slide the needle out with the other hand. Have a transfer pipette available. It can be placed under the catheter to steady, if needed.
  9. Check the position of the catheter in the trachea, and tighten the suture, taking care that the catheter does not slide out as the knot is tightened.
  10. Fill a 10 mL syringe with saline and attach a 21G 1.5 inch needle. With forceps hold the tip of the heart and lift it up to 90o. Locate the pulmonary artery, running vertically down the back side of the heart and off center to the right side. Hold the needle straight up and down. Starting about ¼ of the way down from the top, insert the needle into the heart behind the line of the pulmonary artery. Direct the needle so that the tip just emerges into the right auricle. Perfuse lung with 10 mL of saline. If the position of the needle is correct, the RBCs will be flushed from the lungs, leaving the lungs white. The lung can be perfused a second time if needed.
  11. Fill a 3mL luer lock syringe with dispase that has been warmed to 37oC and attach it to the cannula. Before instilling the dispase, check that the tip of the catheter is visible, as it can be pushed deeper as the syringe is attached. Instill dispase to fully inflate the lung (2.3 mL for post natal day 28 mice and 3 mL for 6-8 week old mice).
  12. Tighten the suture knot to close the trachea. Be careful that the catheter does not slide out, as it is easily pushed out as the knot is closed.
  13. The lungs will be fragile from the instilled dispase. Dissect out the lung as gentle as possible, with the goal of keeping the lung and trachea intact with the thymus and heart attached. Transfer the dissected lung/trachea/thymus/heart complex into a 14 mL polypropylene tube containing 1 mL dispase. Incubate at 37oC for 6 minutes.
  14. Pour the dispase from the polypropylene tube. In a dish containing room temperature saline, excise the lung from the heart and thymus. Separate the individual lobes from the lung. Remove any visible airway tissue and transfer the lobes to a prepared C-tube. Hold at room temperature. Prepare 4-8 lungs to this point, and then continue processing them through the remaining steps.
  15. Add 60 μL of DNAse (10 KU/mL) to the C-tube.  Insert C-tube into the GentleMACs Dissociator, select program m_lung_1.01, and press start.  The program dissociates the lung in 10 seconds. Re-run if the tissue is not fully dissociated.
  16. Incubate lungs at room temperature for 10 minutes.

 

Cell Isolation

 

  1. Centrifuge C-tube at 1500 rpm (433 x g) for 6 minutes at 4o Remove supernatant and combine cells from two mice cell digestions in 2 mL of red cell lysis buffer and incubate on ice for 2 minutes.
  2. Add 8 ml of DMEM media and centrifuge at 1500 rpm for 6 minutes at 4o
  3. Remove supernatant and resuspend in 10ml DMEM and filter cells through 30 μM cell strainer. Wash C-tube with 5 mL of DMEM media and pour onto cell strainer.
  4. Count cells – use a 1:10 dilution. Typical yield: 40-50 million cells per mouse.
  5. For four mice use two columns for CD45+
  6. Centrifuge the conical tubes at 1500 rpm for 6 minutes at 4o Resuspend 107 cells in 90 μL of MACs buffer and add 10 μL CD16/32 to block Fc receptors. Incubate for 10 minutes at 4oC.

      (For higher cell numbers, scale up buffer and antibody volume accordingly)

  1. Then add 10 μL anti-mouse CD45 microbeads per 107 Mix well and incubate at 4°C for 15 minutes.

 (For higher cell numbers, scale up buffer and antibody volume accordingly)

  1. Wash cells by adding 1−2 mL of MACs buffer per 10⁷ cells and centrifuge at 1500 rpm for 6 minutes at 4oC. Aspirate supernatant completely.
  2. Resuspend up to 10⁸ cells in 500 μL of MACs
  3. Place LS Column in the magnetic field of a MACS Separator (orange magnet attached to stand).
  4. Pre-rinse the column with 3 mL of MACs buffer and place 15 mL conical under columns to collect eluent.
  5. Apply the cell suspension (500 μL) onto the pre-separator filter over the rinsed column.
  6. Proceed with three washing steps with 3 mL of MACs buffer each wash and add MACs  buffer only after the previous wash has eluted through the column.
  7. Remove the column and collect the cells with 5mL MACs buffer in a 15 mL collection tube by firmly pushing the plunger into the column and keep as CD45+ cells.
  8. Centrifuge the CD45+ cells at 1500 rpm (433 x g) for 6 minutes at 4o Resuspend cells at a pre-determined concentration and do FACS to determine purity using 1.5 X 105 cells.
  9. Collect total effluent; this is the CD45 fraction and proceed with enrichment of CD326 (EpCAM)+ cells .
  10. Repeat the steps from 23 to 31 with CD326, CD31, and CD140α Microbeads to separate CD326+, CD31+, and CD140α + cells, respectively. At this point it will be possible to combine the preps and use one column.
  11. Centrifuge the CD45+, CD326+, CD31+, and CD140α + cell suspensions, resuspend in complete media (DMEM) and count the cells.

 

 

 

Reagents

 

DMEM media:  Remove 35 mL from 1L bottle of sterile water.  Add powdered media, sodium bicarbonate (3.7 g), 1M HEPES (25 mL) and PenStrep (10 mL).  Mix to dissolve and filter.

 

DNase (Sigma D4527):  Add 28 mL of sterile saline per vial.  Aliquot 500 μL per tube and store at -20°C.

 

Corning (BD Biosciences, BD354235)

 

Red Cell Lysis Buffer (Cat# 00-4333-57, eBiosciences)

 

MACS BSA Stock Solution (Cat # 130‑091‑376, Miltenyi Biotec)

AutoMACS Rinsing Solution (Cat # 130-091-222, Miltenyi Biotec)

LS Columns (Cat# 130-042-401, Miltenyi Biotec)

 

FcR Blocking Reagent, mouse (Cat # 130-092-575, Miltenyi Biotec)

 

CD45 MicroBeads, mouse (Cat # 130 052 301, Miltenyi Biotec)

CD326 (EpCAM) MicroBeads, mouse (Cat # 130 105 958, Miltenyi Biotec )

CD31 MicroBeads, mouse (Cat # 130-097-418, Miltenyi Biotec )

CD140α MicroBeads, mouse (Cat # 130-101-502)

Pre-Separation Filters (30 µm) (Cat # 130-041-407, Miltenyi Biotec )

Ethylenediaminetetraacetic Acid (Cat # BP2482-100, Fisher Scientific)

 

***this protocol is adapted from Messier et al., Experimental Lung Research 2012.

By |2016-11-18T14:14:45+00:00February 1st, 2016|Categories: SOP|0 Comments

CCHMC SOP Postnatal Lung Tissue Fixation

 

  1. Set up and document date of harem breeding.
  2. Day mice are born is recorded as PND 0.
  3. Record date harvested.
  4. Set up the inflation fixation apparatus. The apparatus consists of a ring stand, a clamp, and a 3cc or 10cc syringe barrel. Attach a 23 Gauge butterfly to the syringe barrel with a clamp to be able to stop the flow of PFA through the butterfly. Put the syringe barrel through the ring stand clamp. Fill the syringe barrel with 4% PFA/PBS prepared with DEPC treated water and allow to flow completely through the butterfly. Clamp the butterfly to prevent the flow of 4% PFA/PBS and add 4% PFA to the barrel of the syringe to fill the syringe barrel. Adjust the barrel so that the meniscus of the 4% PFA solution is 25 cm above the trachea of the mouse.
  5. Inject mice with 0.1ml pentobarbital. Wait until mouse is unresponsive by toe pinch.
  6. Spray the abdominal surface with 70% ETOH.
  7. Isolate a small piece of tail for isolating genomic DNA to determine gender if unable to visually determine gender.
  8. Make a small cut with scissors at the pubis followed by a longitudinal cut along the central midline through to the chin.
  9. Dissect away the skin leaving the abdominal wall intact.
  10. Open the abdominal cavity by grasping a small piece of muscle and making an incision.
  11. Exsanguinate mouse by cutting the inferior vena cava
  12. Insert one blade of scissor under the rib cage and cut through the midline while applying upward pressure on the bottom scissor blade.
  13. Dissect away the diaphragm.
  14. Separate the rib cage and remove as much of the rib cage as possible to expose the heart and lungs.
  15. Dissect away the salivary gland with forceps.
  16. Dissect away the muscle around the trachea to fully expose the trachea.
  17. Tie a knot around the trachea with a suture but do not fully close the knot around the trachea.
  18. Make a small incision in the ventral side of the trachea with a pair of scissors and insert the butterfly bevel side up. Carefully close the suture around the trachea and inserted needle. Alternatively, insert the cannula through the mouth/larynx.
  19. Open up the butterfly clamp allowing the 4% PFA/PBS to flow into the lung. Once the lungs are inflated to the correct level of inflation fixation, clamp the butterfly to prevent the flow of 4% PFA/PBS.
  20. Remove the butterfly and completely close the suture knot around the trachea.
  21. Remove the lung, heart and thymus en bloc.
  22. Place the dissected tissue in a 50ml conical tube containing 20ml 4% PFA/PBS.
  23. Place on ice and then place at 4oC overnight.
  24. Following day wash 3X in 1X PBS made with DEPC treated water for 10 minutes each wash at room temperature.
  25. Dissect away the individual lung lobes (Right Upper, Right Middle, Right Lower, Left Lobe, and accessory lobe). Lung lobes (right upper, right lower, and the left lobe) were bisected near the large bronchi with a razor blade. To maximize tissue for frozen and paraffin embedded tissue,  The upper portion of each bisected upper right, lower right, and left lobes and the intact right middle lobe were processed for paraffin embedding as described in step 30. The lower portion of each bisected upper right, lower right, and left lobes and the intact accessory  lobe were processed for OCT embedding as described in step 26.
  26. Place lungs in 30% Sucrose/1X PBS (DEPC treated water).
  27. Incubate lungs O/N in 30% Sucrose @ 4o Alternatively, tissue can be stored in this solution @ 4oC.
  28. Day before freezing place lungs in 2 parts 30% Sucrose and 1 Part OCT and incubate @ 4o
  29. Alternatively, lungs can be prepared for paraffin embedding.
  30. Following step 25, lungs are dehydrated through 30% ETOH, 50% ETOH, and 70% ETOH washes 3X for 10 min each wash. Lungs can then be stored in 70% ETOH at 4oC until processed on tissue processor and then embedded in paraffin. The bisected lobes were embedded with the cut side facing down into wells. (This maximizes the viewing of proximal and distal regions of the lung and extends the amount of tissue available).
By |2015-07-19T23:44:10+00:00July 19th, 2015|Categories: SOP|0 Comments

CCHMC SOP Frozen and Paraffin Sections Antibody Staining

  1. Cut 7 µm sections of frozen tissue on a cryostat and dry overnight at room temperature. Alternatively, 5 µm sections of paraffin embedded tissue can be used.
  2. Look at tissue under a light microscope and make notes of the various structures (i.e. cartilaginous airway, bronchioles, pulmonary arteries, veins, alveoli, etc.) present for each individual slide. This will help determine the appropriate antibodies to use to stain the various structures.
  3. Store at -20oC for short term storage or -80oC for long term storage for frozen sections. Paraffin sections can be stored at 4o
  4. Wash frozen tissue 2X in 1X PBS for 5 minutes each wash to rehydrate and remove OCT from tissue.
  5. Incubate slides in 4% PFA (to refix tissue) in 1X PBS, pH7.4 on tissue for 5 minutes.
  6. Wash 1X with 1X PBS.
  7. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin. Paraffin sections are then placed in xylene 3X for 10 minutes each, followed by 3X in 100% ETOH for 3 minutes each, 95% ETOH for 3 minutes each, and 70% ETOH for 3 minutes each. Slides are then placed in 1X PBS for 5 minutes to completely rehydrate tissue.
  8. If antigen retrieval is necessary to unmask nuclear antigens, antigen retrieval, pH 6.0 (times will vary according to microwave) is performed
  9. Fill plastic coplin jars with Antigen Retrieval buffer (10 mM sodium citrate, pH 6.0). Place in a Tupperware container and fill the Tupperware container half way with dH2O Heat in a microwave according to the instructions on the microwave.  We use a GE microwave for 6.5 minutes at 1100 watts, 6 minutes at 440 watts, and finally 6 minutes at 440 watts. *We usually do a series of three runs to equal the time/temp because of evaporation (refill plastic coplin jars with dH2O).
  10. Cool on countertop, 15 min.
  11. Rinse with dH2O
  12. Wash1X PBS, pH 7.4 for 5 minutes.
  13. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  14. Block for 1-2 hours at room temperature in 4% Serum/PBS/0.2% Triton X-100 with gentle shaking. Typically tissue will be blocked with goat, donkey, or horse serum. The serum species for blocking usually corresponds with the species the secondary antibodies are raised. For example with goat anti-rabbit secondary use goat serum for blocking.
  15. Remove blocking solution and add primary antibodies at pre-determined antibody titers diluted in the appropriate blocking solution. Antibodies are diluted in blocking solution and spun down in a µfuge for 10 minutes at full speed. Apply antibody solution to a cover well incubator (Electron Microscopy Sciences, Catalogue # 70324-20) and invert slide containing tissue onto antibody solution to overlay. Place slides containing antibodies in a humidity chamber and incubate overnight at 4oC.
  16. Wash 3-5X with 1X PBS + 0.2% triton X-100 for 5 minutes with gentle shaking at room temperature.
  17. The appropriate fluorescent secondary antibodies are diluted in blocking solution and spun down in a µfuge for 10 minutes at full speed. Apply the antibodies to the tissue and ncubate in the humidity chamber at room temperature for one hour.
  18. Wash 3-5X with 1X PBS + 0.2% triton X-100 for 5 minutes with gentle shaking at room temperature.
  19. Apply DAPI solution diluted at 1:2000 and incubate at room temp for 15 minutes.
  20. Wash 3-5X with 1X PBS + 0.2% triton X-100 for 5 minutes with gentle shaking at room temperature.
  21. Wash 3X with 1X PB without NaCl or triton X-100.
  22. Flick the slides to remove excess PB and add a drop of Prolong Gold anti-fade mounting medium (Invitrogen, P36930).
  23. Apply a Gold Seal Coverslip (#1.5, Cat# 63792-01 Electron Microscopy Sciences, 24 X 50 mm).
  24. Allow the Prolong Gold to cure overnight in a light sealed box at room temperature and then store the slides in a slide box at 4oC.
  25. Obtain 1024 X 1024 z-stacks of the slides on an inverted Confocal Microscope at various magnifications.
  26. Process the z-stacks in Imaris (Bitplane) imaging software. Export as .tiff files at 600 DPI.
  27. Levels adjustment in Adobe Photoshop can be used to increase the brightness of the image as needed.

Appendix

 

Antigen Retrieval Solution

18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5

82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2

1L dH2O

pH 6.0

By |2016-11-18T14:15:26+00:00February 22nd, 2015|Categories: SOP|0 Comments

CCHMC Mouse SOP Timed Matings and Tissue Harvest, Fixation, and Processing

CCHMC Mouse SOP:  Timed Matings & Tissue Harvest/Fixation/Processing

A.  MICE

1.  C57BL/6J are obtained or derived from the Jackson Laboratory colony.

2.  Breeding females should be between 8-15 weeks of age and no older than 6  months.

3.  Male mice can be used up to 7-8 months of age, or until no longer producing pregnancies.

4.  Expect seasonal changes in breeding performance. Some strains produce more litters in the spring and summer than in winter and fall.

B.   BREEDING

1.     Females should have one litter before setting up breedings for experimental material.  NOTE:  First litters may not be representative of the average number of pups per litter for the strain.

2.     Place 1 to 3 females into each male’s cage in the late afternoon, prior to the dark-cycle. Record date and time.

3.     Check for vaginal plugs early the next morning (plug will dissolve over time).

4.     By convention, the date a plug is observed is designated as gestational day 0 or 0.5.   We use 0.5 (or E0.5).  Record date and time.

5.     Remove male from cage.

6.     Separate unplugged female mice from the male.   Do not use these female mice for breeding until it can be demonstrated that there is no pregnancy.  This can be done by monitoring daily and/or by palpitation.

C.   HARVESTING

1.     Harvest embryos in the morning (before noon) on the desired gestational day.

2.     Obtain fetuses by hysterectomy.  Dam should be anesthetized and then exsanguinated to prevent congestion of blood in the tissues.

3.     Remove pups from uterus and maintain in a large weigh boat on ice.  As the pups are extracted from the uterus, place each pup in a smaller weigh boat on ice, and number or label for future identification.

4.     Strip way extra-fetal membranes.

5.     Weigh and measure crown-to-rump (C-R) length for each pup prior to fixation and record. NOTE: Variations in lung development within a litter are directly correlated with fetal body weight and crown-to-rump length measurements.

6.     Compare body weights and C-R lengths with fetal growth tables to determine developmental age.  See attached or below.

7.     Remove a piece of tail or tissue for isolating genomic DNA.

D.  FIXATION AND PROCESSING

1.     For E12.5 to E14.5, leave embryos intact and immerse in 20 ml of 4% PFA in 1XPBS (formulated with DEPC-treated water) in a 50 ml sterile tissue culture tube.  Fix at room temperature for one hour, and then at 4oC overnight. Number/label tubes accordingly.  NOTE: Volume of fixative should be at least 10-20 times the volume of the tissue.  Container should be at least 2-3 times the diameter of the tissue.  Fixative should cover fetus

2.     Larger pups between E16 and postnatal day 5 should be kept on ice as above, weighed and measured, and then exsanguinated.

3.     Open the abdomen and clip the descending aorta and/or vena cava to let the animal bleed out.

4.     Divide the pup into two segments with a sharp razor blade at the level of the diaphragm, cutting just below the inferior extent of the thorax or below the liver.

·      Remove liver and diaphragm, and trim rib cage.  NOTE:  You must strip off the diaphragm prior to immersion in the fixative in order to allow the fixative to enter the chest cavity directly.

·      If leaving the chest intact, the head may be removed by bisecting it between the upper and lower jaws.  The lower jaw will still be attached to the neck.  This prevents the larynx and trachea from retracting.

·      Alternately, remove lung from the chest.

·      Place in 20 ml of 4% PFA/1X PBS, incubate at room temperature for 1 hour, and then incubate at 4oC overnight.  Number/label containers accordingly.  NOTE: If the fixative becomes bloody from the initial dissection, then rinse and replace with fresh fixative.

3.     Wash 3 times in 1X PBS (formulated with DEPC-treated water) for 10 minutes each at room temperature.

4.     Incubate in 30% Sucrose in 1X PBS (formulated with DEPC-treated water) at 4oC.

5.     The day before freezing place in 2 parts 30% Sucrose/1XPBS and 1 part OCT, and incubate at 4oC.

6.     Freeze in molds filled with OCT on dry ice or in isopentane that has been pre-chilled in a container surrounded by dry ice.

7.     Alternately, embryos can be prepared for paraffin embedding.  Following step 3, the tissue is dehydrated through 30% and 50% ETOH for 10 minutes each, and then 70% ETOH, 3 times, 10 minutes each.  All solutions are prepared with DEPC-treated water.  Tissue can then be stored in 70% ETOH at 4oC until processed for paraffin embedding.

8.     Embed each fetus so that it is resting on its back with the heart facing up.  When embedding E18.5, lift up the fetus so the fetus is resting more on the shoulder blade.

E.  RNA EXTRACTION

1.     Alternatively, tissue can be harvested for RNA extraction. Extract the lungs from the chest and place in 1X PBS.

2.     Carefully, dissect each lobe away from the heart, trachea, bronchi, esophagus.

3.     Place in collection tube and immediately place on dry ice. Store at -80oC until RNA can be isolated.

F.  SOLUTIONS FOR FIXATION / PROCESSING

1.         PBS (phosphate-buffered saline)

10X stock solution, 1 liter, pH 7.2 to 7.4*         20X stock solution

80       g          NaCL                                             160 g     NaCl

2          g          KCl                                                     4 g    KCL

11.5    g          Na2HPO4-7H2O                                23 g    Na2HPO4-7H2O

2          g          KH2PO4                                              4 g    KH2PO4

2.         4% paraformaldehyde in PBS

Must be made up fresh, i.e. on the same day as fixation.

(a)       Recipe #1 – for small volumes.

10       ml of 16% paraformaldehyde stock solution, EM grade*

4          ml of 10X PBS (prepared as above)

26      ml of D2H2O

40       ml total (pH 7.2-7.4)

(b)      Recipe #2 – for larger volumes.

Add 8 g to 100 ml of water (ddH2O).  Heat to 60°C in a fume hood.  Add a few drops of 1 N NaOH to help dissolve.  When the solid has completely dissolved, let the solution cool to room temperature, and add 100 ml of 2X PBS and adjust pH to 7.4.  This solution should be prepared fresh.  You may have to filter solution if a precipitate persists.  We have also frozen batches of this solution at -20°C for subsequent use.

These are basic recipes.  You will have to make more if fixing a large number of animals.  Figure 20 ml per fetus (or 10X the volume of the specimen).  You may fix the tissue in sterile 50 ml tissue culture tubes.

*obtain from Electron Microscopy Sciences, #15710,10 X 10 ml per box, 1-800-523-5874.

G.   Gender Identification PCR

http://www.karger.com/Article/FullText/348677

McFarlane L, Truong V, Palmer J, S, Wilhelm D, Novel PCR Assay for Determining the Genetic Sex of Mice. Sex Dev 2013;7:207-211

SXF  5ʹ-GATGATTTGAGTGGAAATGTGAGGTA-3ʹ

SXR, 5ʹ-CTTATGTTTATAGGCATGCACCATGTA-3ʹ

PCR Conditions

94oC     2 minutes

94oC

57oC                   30 Seconds, 35 cycles

72oC

72oC   5 minutes

4oC     Hold

By |2016-11-18T14:15:29+00:00February 3rd, 2015|Categories: SOP|Tags: , , , , , |0 Comments