Mice
References: “Anatomy of Mouse”
Mice are the mainstay of in vivo immunological experimentation and in many respects they mirror human biology remarkably well. This conservation of function is reflected on the sequencing of both the human and mice genomes, which reveal that to date only 300 or so genes appear to be unique to one species or the other. Despite this conservation there exist significant differences between mice and human in immune system development, activation, and response to challenge, in both the innate and adaptive arms. (Mestas, “of Mice and Not Men: Differences between Mouse and Human Immunology” J Immunol 2004, 172, 2731-2738)
Differences between Mice & Humans:
Murine models have been extensively used to identify and test drug candidates for subsequent human trials. However, few of these human trials have shown sucess. The success rate is even worse for those trials in the field of inflammation. Although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate pooly with the human conditions and also, one another (seok, “Genomic responses in mouse models poorly mimic human inflammatory diseases” PNAS, 2013).
The balance of lymphocytes and neutrophils in adult animals is quite different: human blood is neutrophil rich (50-70% neutrophiles, 30-50% lymphocytes) whereas mouse blood has a strong preponderance of lymphocytes (75-90% lymphocytes, 10-25% neutorphils). (Mestas, “of Mice and Not Men: Differences between Mouse and Human Immunology” J Immunol 2004, 172, 2731-2738)
Mouse Models for Particular Diseases
–For inflammatory diseases: Despite the artifical way of inducing inflammatory responses, animal models of disease have proven invaluable for providing insight into the potential efficacy of new drugs, particularly when careful consideration has been given to ensure that the model system under study resemles the inflammatory pathway expected in human disease. The most common artifical approaches for sitmulating inflammatory diseases in mice are quite varied, and range from overexpression or targeted deletion of genes in transgenic or knockout animals, immunization of animals with putative autoantigens, to synthetic, chemical challenges (Heather, Advanced Drug Delivery Reviews 59 (2007) 1084-1092.
The three most coommonly used modes for RA are adjuvant-induced arthritis (AIA) in rats and collagen-induced arthritis (CIA) in rats and mice (Hegen, Ann Rheum Dis, 2008, 67, 1505-1515. Important cirteria in selection of a model include 10 capacity to predict efficacy of agents in humans, 2) ease of performing the model, reproducibility of data, reasonable duration of test period and 3) similar pathology and/or pathogenesis to that of human disease. (Bendele, Animal models of rheumatoid arthritis, J Musculoskel Neuron Interact 2001, 1(4), 377-385.
Types of Injections:
Experimental immunogens are usually administered parenterally (para- around, enteric-gut) which means by routes other than the digestive tract. Antigen administered intravenously is carried first to the spleen, whereas it moves first to cocal lymph nodes if injected subcutaneously. The following are the most common routes:
(A) Intraperitoneal injection (ip): (into the peritoneal cavity) This is an injection to the stomach area of the mouse.
(1) Obtain syringe without needle
(2) grab behind head of mouse. Let mouse pull cage. Inject needle into peritoneal cavity.
(B) Intravenous injection (iv): is intravenous into a vein (like the tail vein)
(1) Place mouse in tube that has opening for tail.
(2) fill up your syringe and then put on needle using cap. Tighten needles head. The tail vein is the only vein assessible in the mouse. The vein should be heated up before injection.
(3) Pad tail with alcohol swab
(4) keep needles horizontal and inject into tail vein, then squirt out volume. (never let needle touch any surfaces! you can keep it pronged up on the cap!) Bending the tail with your free hand using your forefinger while you inject will help you control the tail.
(C) subcutaneous injection (sc): This is an injection beneath the skin
Tail Bleeding:
Need a sharp instrument like a scaple. Use a pipet to get the blood.
Euthanizing Mice:
CO2 is one way that mice are euthanized. Place paper down in chamber first. Also place some paper under the chamber (use the thick white paper). Do not prefill chamber since mice can sense danger. Always right down the date of birth, type and sex of mice used. After use, wash cage with water and then again with distilled water (do not use alcohol with plastic box. put chamber tilted on paper towels to dry)
Obtaining Thioglycalated Macrophages from Mice
(1) Transfer 3 ml of thioglycolated broth to a 50 tube. Let stand at room temp to warm up
(2) obtain 21G 1/2 gauge and 25G 3/8 needles + 3 ml syringe
(3) use the 21 1/2 needle to take up the 3 ml of broth. (squirt out a little bit after sucking up to make sure all air bubbles are out.
(4) Go to room 1336, select mouse, mark tail with black marker. Inject all 3 ml into peritoneal cavity (inject in between the nipples of the mouse). (hold tail with hand and grab fur behind head) Proceed next to obtaining the peritoneal macrophages
____________________Obtaining the peritoneal macrophages___________________
(1) After 4 days, pick up new cage and place mouse in cage. Note date received mice and add # of weeks since then to obtain the age of mice (ie., 7 weeks old at 2/10/04 but you do the experiment on 3/1/04, mice are 7 weeks 10 days)
(2) Prepare 1% PBS
(3) Obtain bucket of ice (will need this to keep your cells on it) and 21 1/2 G syring and 5 ml syringe
(4) euthanize mouse, cut fir in center with flame steril scissors, pull back fur (half front, half back)
(5) Inject 5 ml of the PBS into peritoneal cavity (just under membrane, avoid organs)
(6) shake mouse gently and suck out about 5 ml with same needle and transfer to 50 ml tube
(7) transfer the rest of the PBS to your 50 ml tube
(8) spin at 10C, 10 min 1100 RPM, remove supernatant
(9) (resuspend cell in 1 ml 10% RPMI. (take 50 ul of this + 50 ul tryptan blue) (ex. you count 162 live cells. so 162 x 5 x 2 x 104 x 1 = 162 x 106. This, however, is not a pure population of macrophages. So divide this # by 2. so 8.1 X 106 cells or about 8.0 X 106 cells/ml.
(10) Transfer cells to a 96 well culture flat bottom plate (you might put say 120 ul of cells per well, 0.5 ml or 1 ml depending on how many cells you have to work with. But this concentration is typically at 1.0 X 106cells/ml)
q. What volume should you transfer from your 8.0 X 106 cells/ml to each of the well plates?
One way you could do this is to bring your 8.0 X 106 cells/ml up in a total volume of 8.0 ml (so would need to add 7 ml) so you have 1.0 X 106 cells/ml. You could then add 1 ml from this tube to each of your culture wells. But this would leave you only 8 wells to do what ever it is you want to test. Suppose from the outline of your experiment, you determine you will need to use 13 culture wells in a 24 well plate. What you would have to do in this case is to distribute a smaller volume. if you were to have a total volume of 0.5 ml, increase the # of wells you could use from 8 to 16. Your [cells] would not change, it would still be at 1.0 X 106 cells/ml (you would simply now have 0.5 X 106 cells/ 0.5 ml which is the same as 1.0 X 106 cells/ml.
(11) After an hour time of incubation at 37C, wash the cells. (Using themulti channel pipette pipette up and down and discard all the liquid (this does not contain MQ, remember those are at the bottom of the plate) into a trough. Put new media into another trough and add same volume (0.5 ml) to the wells. pipette up and down and discard. Do this 1 more time for 1 total of 3 times and discard.
(12) Now add media to your wells. (remember here that the total volume can not change. It must remain at 0.5 ml. So for your cells only wells, you can add 0.5 ml media. But if you calculated that you are going to add say 5 ul of LPS, you would need to add 495 ul of media, then 5 ul LPS. If you were going to add 4ul LPS + 5 ul EGC, you would need to add 490 ul media + 5 ul LPS + 5 ul EGC.
Obtaining Spleenocytes from Mice
(1) place 12 ml HBSS solution into bag and keep it on ice.
(2) Spleen is on left side of animal. So place mouse on side so left side is facing you. cut peritoneal membrane of mouse
(3) locate spleen (right side) and using flame sterile tweezers and small scissors, cut around the this membrane. Cut spleen at two points and lift out.
(4) place spleen in bag with your 12 ml hanks.
(5) place bag into stomacher machine (flap down outside machine) and use light for several seconds
(6) transfer contents of bag into (15 ml) tube using glass pipette (avoid any tissue particles)
(7) spin
(8) transfer ACK into tube to lyse red blood cells
Obtaining Dendritic Cells from Mice
Reagents
a) prepare 100 ml filter sterilized 10% RPMI complete and 100 ml flushing media (99 ml + 1 ml antibiotic)
b) place ice in bucket. obtain 2 petri dishes, large scissors, forcepts, paper white squares (cut one towel into square sizes) and 2 plastic bags (1 small, 1 large)
c) take keys from drawer (the one w/ JJJ opens door downstairs)
d) euthanize mice (see above)
Obtain the Hind Legs
1) Before cutting the mouse, squirt ETOH (70%) onto stomach area. [70% ETOH can be made by adding 70 Alcohol and 20 ml water or 150 alcohol and 40 ml water]
2) cut small incision and pull skin back (part to head and part to feet)
3) remove skin past feet. Then remove feet (do not cut too close to leg or will rupture)
4) Cut as close to spinal cord as possible
5) place legs into petri dish (2 filled with cold PBS (do this under the hood) 1 dish is for the bonds and 1 for bones) which is placed on ice.
6) place mouse remains in separate paper bag and soiled paper in separate larger plastic bag.
Remove muscle tissue
1) using side of forcepts to tear tissue first then use sides to push tissue off bone
2) cut off any hind leg portions
3) separate femor bone from tibia
4) place bones in 2nd plate with PBS
Sterilize the Bones
1) For flushing solution (just RPMI + antibiotic) ( Prepare about 200 ml unless some is made already; ex. 1 ml antibiotic + 99 ml RPMI, or 20 ml + .2 ml ant) (Filter Sterilize!)
2) obtain about 3 50 ml tubes, a surgical blade, 4 petri dishes, 1 syringe (3 ml) and 2 needles (smaller blue (which is usually attached to the 3 ml syringe, and larger green (25 1/2 needle) used to suck up the medium.
3) Label plates. In first petri dish pour ETOH 70%. In second-4th dishes pour cold PBS.
4) place the bones 1st in the dish with ETOH for 2 min (use timer).
Wash the Bones
1) shuffle the bones from plates 2-4 which have the cold sterile (needs tape, otherwise filter) PBS. (the PBS is to remove the alcohol from bones).
Flush the Bones
1) pour flushing media from jar into 50 ml Tube
2) take up about 3 ml of flushing media with larger syringe needle (21 G 1 1/2), then transfer to smaller needle (25 5/8).
3) transfer bones (one at a time) using forcepts to an empty petri dish and use scalple to cut the ends of the bone (cut just enough to open cavity)
4) use another petri dish to capture the flushing solution. Insert the smaller needle into one end of the bone and flush out the cells into that dish. (move needle slightly up and down as you flush the cells) (once petri dish starts to get full, transfer the cells to a 50 ml tube)
5) make sure you have all of your cells in the petri dish transferred to 50 ml tubes. Equally distribute the solution in these tubes (add more media if you need to)
6) centrifuge 10 min at 10 C at 1100 RPM
7) clean area (put sharps in sharps container) wash surgical blade holder and forceps with hot water and place on paper towel.
8) remove supernatant from tubes (should see red pellet at bottom)
Lyse Red Blood Cells
1) add 3 ml ACK into each tube with pellet (pepette up and down to resuspend)
2) after about 30 sec, add about 12 ml HBSS to stop lysing (or can just add up to 40 ml on the 50 ml tube)
3) repeat (1) with any additional tubes having a pellet
4) spin tubes down again.
5) remove supernatent (now should see white pellet)
Count Cells
1) Resuspend the cells in a convenient amount of Hanks (e.g., 10 ml Hanks)
2) take sample to count the cells
3) centrifuge the tube again 15 ml tube (10 min)
4) count the cells while they are spinning down.
Culture the Cells
1) Pour off the supernatant. We want to resuspend our cells at a concentration of 1 million cells/1ml medium. Suppose that we calculate from counting that we have 40 million cells. What volume should we add to our pellet to get this?
to make it easy just add 40 ml.
2) Add GM-CSF (this is a cytokine which which encourages dendritic cells to stay immature and is in the 2nd shelf up from MDP) Get stock vial marked 600 ng in freezer. Add 1 ml to the via. You want a final concentration of 5 ng/ml.
q. How much GMSF should u add?
(600 ng/ml)V1=(5 ng/ml)(26 ml)
V1=216 ul
3) Obtain necessary amount of 6 well plates. Disperse 3 ml of cells into each well of each plate. Cross out wells not used
q. you have 26 ml total volume. How many 6 well plates will you need?
6 wells per plate X 3 = 18. So if have 26 ml total going to need 2 plates)
Changing Media of Cells (Day 2)
24 hours after your plates have be at 37C, you should add fresh medium (10% complete RPMI).
(1) prepare new medium with GMCF ( 5 ng/ml) See caculations above.
(2) Remove supernatent from plate wells using pipete and place into beaker. Pipet up and down to wash well as you transfer. The cells we are interested in are adherant so you can discard the transferred media here down the drain.
(3) add 3 ml of new media to each well (As a precaution you can also filter your media. Obtain filter (size pours .22 um), put filter on flask, pour media into top of filter)
(4) reincubate at 37 for 2 more days.
Changing Media of Cells (Day 5)
(1) several days later you will need to change the media again. This time simply withdraw 1 ml of media from each culture well and replace it with 1 ml of new media (+GMSF 5 ng/ml). So make up enough media to add 1 ml to each well. Calculations of GMSF are as above.
Ex. 600 ng/ml = (5ng/ml)(10ml)
v1=83ul
Dissecting Mice:
1) turn on gas and burner 2) using scissors which has been dipped in 70% alcohol 3) cut skin in center and pull skin back. 4) removed wanted organs (ie., spleen, or lymph nodes (on either side of arm pits, or mesenteric which are near intestines) and place into 12 ml hanks medium (balanced salt solution) which is in special stomacher bag. 5) use stomacher (10 sec, high) to break up spleen.
Infection of Mice
In this exeperiment we will infect mice with Legionella (Lp) which was grown in a plate overnight. The infections will be done at 6 hour, 2 hours and 3 hours. Since using a biohazardous material, always keep blower on. Place cages in containment cage (which should say “Negative” pressure). Place mouse parts into separate red auoclave bag and tie it. Sharps go into special sharps container. Turn uv light on at end of experiment. Place any cages you have finished housing the LP mice into red autoclavable bag.
(1) Obtain and label plastic bags in which you will be placing your spleens, lymph nodes
(2) Do OD reading on your LP culture. Add some pyrogen free saline to 15 ml tube. Using cotton swab, roll in plate and swirl end in tube containing saline. vortex Repeat with 2nd swab end. Use car with writing on back. You should be able to just make out the letters through the solution.
(3) Transfer 0.8 ml from 15 ml tube to disposable cuvette. Take OD reading. (Use chart to convert) This will tell you how many colony forming units (CFU) you have.
(4) obtain syringes. Set up heat lamp. Work under hood. Obtain another cage that you can put mice in to heat up. Get yellow cards from office and label properly.
(5) Do tail vein injections (see above)
(6) Prepare bags with 12 ml Hanks solution and label the bags appropriately. (ex. “spleens.”)
(7) obtain 25 gauge syringe. Take needle head off and pull up necessary volume of heparin. Pull up about 100 ul of the heparin per needle for every mouse that you will use. Put needle head back onto syringe and then pull the heparin into the syringe back and forth a couple of times to coat the entire syringe. Push out just a small amount of heparin through the needle.
[Herparen is necessary so that blood does not clot. Here, we will use 1000 units of heparin. So label 15 ml tube “hearin 1000 u/ml. The vile we use says “10,000 units/ml so we need to dilute this 1:10 to get 1,000 units/ml. so add 1 ml of the stock with 9 ml PBS)
(8) 1 hour post infection, euthanize mice with CO2 (see above).
(9) Using syringe perform cardiac punch. In this procedure, you will be taking blood up into syringe. Use your fingers to message the blood out into syringe after you poke the heart cavity. Eject blood from syringe into 15 ml tube
(10) Perform dissection and remove lymph nodes (periferal as well as messenteric) (see above). Place into your bags.
(11) Grind the organs in each bag using stomach grinder. draw volume from each bag you used with pipette and eject into 15 ml tube. Centrifuge tubes
(12) pour off the supernatent (the cells will be in the pellet)
(13) ACK the lymph nodes (because want to culture them) Put PBS into conical tube having the spleen and place mixture into small 1.5 tubes. Resuspend into Trip reagent. Put in box, label and place at -80.