MOUSE :: HMDP :: Peritoneal macrophages, inflammatory responses

G.         Hybrid Mouse Diversity Panel:  Peritoneal macrophages, inflammatory responses

1.         Mice (L. Orozco)
2.         Peritoneal macrophages, isolation, treatment (L. Orozco)
3.         Expression arrays, control, LPS, OxPAPC and diesel exhaust treatment (L. Orozco)
4.         Pilot studies (L. Orozco)
5.         Acetylated LDL uptake (L. Orozco)

 

 

1.         Mice (L. Orozco)

Male mice from 92 of the HMDP inbred strains were obtained from the Jackson Laboratories (Bar Harbor, ME) at 6-10 weeks of age to ensure acclimatization to a common environment. Mice were fed a chow diet (Ralston-Purina Co., St. Louis, MO) and housed until 16-weeks of age, then fasted overnight for 16 hours prior to euthanasia under Isoflurane anesthesia (Phoenix Pharmaceutical Inc., St Joseph, MO). All mice were housed under pathogen-free conditions and according to NIH guidelines.

 


2.         Peritoneal macrophages, isolation, treatment (L. Orozco)

Macrophage culture conditions.  Primary macrophages were harvested from mice by intraperitoneallavage with 1xPBS, four days after injection with 1.5ml of 3% thioglycollate (BD, Sparks, MD). All mice were injected with the same batch of thioglycollate. Cells from different mice of the same strain were pooled and plated in 20% FBS DMEM with 1% penicillin-streptomycin at 4 million cells per well in 6-well plates, and stored in a 37˚C and 5% CO2 incubator. Cells were washed once with PBS the day after plating and incubated for 4 hours with 1%FBS DMEM media in control-treated cells, with control media plus 2ng/mL LPS (List Biological Inc., Campbell, CA, 201), or with control media plus 50μg/mL OxPAPC. OxPAPC was prepared from PAPC (Avanti Polar Lipids, Alabaster, AL) as previously described.1After treatment, media was removed and cells were washed once with PBS.

MACROPHAGE RECRUITMENT WITH THIOGYCOLLATE
Purpose:
In this experiment, we will stimulate macrophage recruitment in mice by injecting a thioglycollate solution into the intraperitoneal cavity. We will collect the macrophages produced by the mouse 4 days after injection and use this to culture the cells from each mouse. The macrophages obtained rom this experiment should be used within 1-3 days after harvest.

Materials
* Thioglycolate solution (4% Thioglycolate):

  • Obtain Brewer Thioglycollate Medium (BD#211716, Lot 3308383)
  • Reconstitute at 37.5g/L of distilled or deionized water. Boil and stir until completely dissolved. Autoclave at leas 1 day previous day of use. Store at 15-30°C; pH 7.2 ± 0.2 at 25°C.

* Sterile troughs/Reagent reservoir (Corning Costar Incorporated Cat. No. 4871)
* 3mL sterile syringes – 1 per mouse.
* Needles: 27 gauge – 1 per mouse.
* Isolflurane anesthesia.
* 70% Ethanol for sterilizing.
* Mice: Record all information on mice injected:

Protocol

  1. Fill syringes with thioglycollate (Do this inside the tissue culture hood)
  • Open thioglycollate bottle, inside the tissue culture hood only (autoclaved). Pour some thioglycollate into a clean, sterile trough.
  • Insert needle into syringe.
  • Uncap needle, making sure not to anything to it, as this part will touch the liquid. Needle and thioglycollate must remain sterile.
  • Fill syringe to a level of 2.00ml, by carefully dipping the needle into the thioglycollate in the trough. Fill syringe until you reach level.
  • Holding syringe straight up, tap the side of syringe to make any bubbles go to center top. Expel a small amount of liquid to get rid or bubbles.
  • Cap syringe.
  1. Inject mice
  • Uncap needle by holding down syringe horizontally, and lightly uncapping needle. Leave this ready on the clean bench. Make sure there are no bubbles in syringe.
  • Obtain mouse from cage.
  • Anesthetize with a small amount of isoflurane…you don’t want to kill the mouse, do not leave mouse too long in the anesthetizing chamber.
  • Restrain mouse by gently, but firmly, pulling skin in the back of the neck. Hold skin between your thumb and the lower part of your index finger. Hold body of mouse in the palm of your hand, and pull the tail between and behind your pinky finger. On left hand.
  • Obtain syringe with right hand, releasing cap. Inject syringe horizontally, with hole side down, directly into the intraperitoneal cavity (inject in lower abdomen).
  • Inject thioglycollate solution gently, using your thumb to push needle pump.
  • Inject until you reach 0.5ml level, so you inject 1.5ml of 4%Thioglycollate.
  • Make sure mouse is ok (regians conciousness) when you put it back in the cage.
  1. Stimulation for 4 days
  • Leave mice for another 4 days to allow macrophage stimulation by thioglycollate in intraperitoneal cavity.
  • Do not leave mice for longer than four days, or you will start getting lymphocytes as well.
  • Do not leave for less than four days, or you will not get as many macrophages.
  • Collect macrophages on the day after fourth day.

MACROPHAGE EXTRACTION: INTRAPERITONEAL LAVAGE
MATERIALS:

  • 10ml sterile syringes (one for each mouse)
  • 20 gauge needles (one for each mouse)
  • Phosphate Buffered Saline, pH 7.2: PBS 1X (GibcoBRL Cat. No. 20012-027)
  • Isoflurane for anesthetizing
  • 70% Ethanol for spraying
  • Instrumentation, autoclaved: 2 surgical scissors, forceps, curled fine forceps
  • 50ml conical tubes, sterile (one for each mouse genotype)
  • Macrophage media: 20% FBS in DMEM high-glucose + penicillin:
  • 50ml Fetal Bovine Serum (ATCC Cat. No. 30-2020), thaw at 37°C before adding (20%FBS)
  • 2.5ml Penicillin-Streptomycin (Invitrogen 15140-122), thaw at 37°C before adding (1%)
  • 197.5ml DMEM high-glucose, +L-glu, no Na-pyruvate (GibcoBRL Cat. No. 11965-092). Thaw at 37°C before adding
  • Combine all and filter. Store at 4°C.

Note: Can use FBS or Pen-Strep from any company, as long as it has been kept sterile and you use it consistently throughout the experiment.

  • Ice bucket
  • 6-well plates for tissue culture (or whatever plate time you are using)
  • Mice:
  • All procedures will be performed in the tissue culture hoods, following tissue culture preventive procedures
  • Mice injected with 1.5ml of 4% Thioglycollate 4 days prior.
  • ACK Lysis Buffer (total volume: 500 ml) 4.15g (0.15M) NH4Cl (Sigma A-0171 100gm), 0.5 g (0.0061M) KHCO3 (Sigma P-9144, 500gm), 18.6 mg Na2EDTA (Sigma ED2SS 50gm), add 400ml H2O and adjust pH to 7.2-7.4 (usually pH is already at 7.3), fill to 500ml, then filter through 0.2 (m cellulose acetate) FILTER. This solution can be kept for months at 4oC.

PROTOCOL
Macrophage Extraction

  1. Obtain all materials. Autoclave instruments. Prepare media as specified and keep at 37°C. Obtain mice.
  2. Fill 10ml syringes with sterile PBS 1X and cap with 20 gauge needles. Mark each with mouse number, genotype and identification. Place on ice. Note: You can aliquot PBS into a 50ml falcon tube when filling syringes, so there is less change of contamination of entire PBS bottle. Fill syringes from 50ml falcon tube.
  3. Anesthetize mouse using isoflurane and euthanize by cervical dislocation
  4. Euthanize mouse by cervical dislocation.

5. Sterilize mouse
- Place mouse in a beaker with 70% ethanol. Or spray mouse with 70% ethanol.
- Place mouse in tissue culture hood immediately.
6. Laporotomy incision
- Make a small incision in abdominal skin of mouse (use one set of forceps and scissors).
- Open up mouse by pulling skin with both hands to each side of mouse. Do this firmly so that skin is completely pulled down, but gently so that you do not open up intraperitoneal cavity or break any limbs. Spray peritoneum with 70% Ethanol.
7. Inject Cold PBS
-  Uncap needle inside the hood and squirt out a small amount of PBS to get rid of any bubbles.
- Inject needle, hole-side down, into the mouse in lower abdominal area, preferably in a region where there is fat.
- Insert needle gently and not too deeply so you do not perforate any organs (it is important not to get blood in the IP cavity).
- Immediately after inserting the needle, begin injecting PBS into IP cavity forcefully.
- Take needle out slowly so that you allow fat to clog the hole (if you take it out too fast, the PBS will come out of the hole).
8. Wash mouse
- Hold mouse by tail and swish around to wash (lavage) IP cavity with the PBS that has been injected.
9. Macrophage extraction
- Using same syringe, go to upper part of abdomen and insert needle horizontally.
- Make sure you do not perforate any organs.
- Important: Hold needle inside peritoneal cavity in a horizontal position, with the needle hole-side down, and extract the PBS containing macrophages. Do this slowly; if you do this too fast, you will aspirate fat and clog needle.
- Try to get at least 7ml of fluid.

* Repeat this procedure for each mouse.

Plating

  1. Collect cells
  • Pour cell suspension in 50ml collection tubes.
  • You may pool suspensions of different mice with same genotype.
  • Centrifuge cells at 1Krpm for 10 min. Make sure you balance the centrifuge.
  • Discard supernatant: Pour off supernatant inside tissue culture hood so you keep only pellet. Discard into another sterile tube, in case you loose pellet while pouring off.
  1. ACK Red Blood Lysis (Optional)

- Re-suspend pellet in 10ml ACK Lysis Buffer and incubate on ice for 5-10 minutes.
- Pellet again under same conditions (1k for 10 min).
- Discard supernatant (pellet should be clean), and resuspend pellet in 10ml PBS.
- Pellet again and discard supernatant.
Note: Do this only if you see  blood in pellet. The least you handle the cells, the better.

  1. Re-suspend cells
  • Using 10ml pipette, add Macrophage media (previously prepared) to the pellet: approximately 3ml per mouse.
  • Re-suspend cells by pipetting up and down. First add 1mL or media using a  p1000 and resuspend, then add the rest of the media using a 5 or 10ml pipet. Be careful not to aspirate all the way up or you will touch filter and contaminate the cells. Watch the pipette and do this very carefully, several times, until pellet is complete dissolved.

Plating Counts: Determining cell concentration in macrophage cell suspensions

  1. Prepare dilutions
  • Add 20μl of cell suspension to 80μl of cold PBS (5X dilution).
  • Take 10μl of this­ 5x dilution + 10μl of Trypan blue dye (2X dilution).
  • Use 10μl of this 10X dilution and pipette into counting chamber.
  1. Counting
  • Count number of cells in each square of the chamber.
  • Average the number for all 4 squares in chamber.
  • Use this number to determine [Macrophages]:

[Macrophages] =     # Average of cells x 10 (DF) x 1x104 = _____________cells/ml

  1. Dilute cell suspension
  • You will dilute cell suspension to obtain a concentration of 4 million cells/2ml, which is 2x106 cells/ml, which will give 4 million cells per well when 2ml of this suspension is plated. Note: You plate this many cells because a lot of them are not macrophages. Those that are not macrophages will not attach, and will be washed off after you plate.
  • Note: For 96-well plates, use 300,000 cells/well, or 150uL of this suspension
  • Determine M1 by counting procedure under microscope: M1
  • Determine V1, this is the actual amount of suspension you have in the tube: V1 = x
  • Perform calculations as specified bellow, for each group.
  • Add the amount of diluent of macrophage media to achieve the desired concentrations:

      V2 - V1 = diluent = y.

  • Dilute: Dilute cells in 20%FBS Media prepared. Re-suspend pellet very well, as before.
  • This will give you a cell suspension with your desired cell concentration.
  • Do this for each pool of cells that you have.

Genotype/Strain: ________

M1 = _?Plug in______________ cells/ml            M2 = 2.0x106 cells/ml

V1 = ?Plug in      _______ ml                                            V2 = _____________ ml

M1V1=M2V2

DFM1  =          M1              =   V2  = x + y
          M2       (2x106 cells/ml)    V1            x

 

?M1                             = x + y
(2.0x106 cells/ml )   x

à                          y =         x* M1                  - x
                                                         (2.0x106 cells/ml)     

Plating

  • Plate 2ml of per well
  • You will add 2ml of the cell suspension at 4 million cells/2ml into each well of the 6-well plate until all cell suspension has been plated.
  • Incubate at 37°C and 5% CO2  overnight.
  • Wash Cells Next Day
  • Aspirate media carefully from each well. Add 2ml of warm or RT (room temp) 1X PBS to each well.
  • Aspirate PBS.
  • Add 2ml of warm 20%FBS media to each well, or,add 1%FBS +/- treatment as treatmet media.

Note: Do one plate at a time, you should not cells dry up.

Lipid Uptake using Di-I-AcLDL
Treat Cells

  1. Harvest mouse peritoneal macrophages and plate 300,000 cells/well in 96-well plates. Plates should be dark-well TC plates (Costar 3603, Fisher 07-200-565). Do not use clear or white plates. Allow cells to attach and settle overnight.
  2. Wash cells with PBS 1X the next day.
  3. Add treatment media to each well using 100uL/well:
    1. Control :                        no treatment              (Biomedical Technologies BT-902)
    2. DiI AcLDL:                  10ug/mL DiI-AcLDL (Biomedical Technologies BT-906)
    3. AcLDL +DiI-AcLDL:  10ug/mL DiI-AcLDL + 200ug/mL AcLDL (unlabeled)

Note: Make sure you do not vortex these solutions, and that they are stored at 4C but not lower, since AcLDL will clump if frozen or if you place the vials on ice.

  1. Incubate cells for 4hours, 37C, in the dark.
  2. After incubation time, follow steps below to read fluorescence. Make sure you turn on fluorometer at least 30 minutes before you plan to use it, and that you have all materials ready.

Read fluorescence
1. Wash cells – After incubation, remove treatment media and wash cells with :
3x with 1xPBS
2. Add 50µL PBS to each well (96-well plates)
3. Read DiI fluorescence (no fixation):        Ex/Em           530/590
                       
Nuclear staining
1. Remove PBS using multi-channel pipet.
2. Fix cells – Add 100uL  4% Para-formaldehyde (PFA at 37˚C). Incubate RT for 10 minues.
3. Wash cells 1x with 100uL PBS, using multi-channel pipet.
4. Stain with nuclear dye: DAPI for the BMS plate (Poly-D-Lys coated) and Sytox for regular black plates
            DAPI              5,000x in PBS             100µL/well                  10 min
(3µM final; DAPI, Molecular Probes D-1306)
or
SytoxGreen     5,000x in PBS             100µL/well                  10min
(1µM final, 5mM stock)
5. Wash 3x with PBS—Remove staining solution and wash 2x with PBS, RT, using multi-channel pipet.
6. Add 50µL PBS to each well.
7. Read fluorescence of nuclear dye and DiI (fixed).
                        Excitation max                       Emission max
DiI                               530                                          590

DAPI                          358                                          461

SytoxGreen                485                                          530

Note: Read first in the SpectraMax plate reader, then in Jesus’ plate-reader.

 


3.         Expression arrays:  control, LPS, OxPAPC and diesel exhaust treatment (L. Orozco)

Total RNA was extracted from macrophage samples using RNeasy columns (Qiagen, Valencia, CA), using on-column DNase digestion (Qiagen) and according to manufacturer’s instructions. Because sufficient RNA for expression profiling was not obtained from all samples, we profiled 86 strains from Control-treated cells, 89 strains from LPS-treated cells and 80 strains from OxPAPC-treated cells. Samples were arrayed using Affymetrix Mouse Genome HT_MG-430A arrays as previously described.2The image data was processed using the Robust Multichip Average (RMA) method to determine the hybridization signal for each gene. To avoid detecting variation signal intensity as an artifact of SNP variation in transcripts, prior to RMA normalization we excluded individual probes in probe-sets that aligned to sequences with SNPs, and excluded entire probe-sets where at least 8 of the probes in the set aligned to a sequence with SNPs.

 


5.         Pilot studies (L. Orozco)

In development.

 


6.         Acetylated LDL uptake (L. Orozco)

Treat Cells

  1. Harvest mouse peritoneal macrophages and plate 300,000 cells/well in 96-well plates. Plates should be dark-well TC plates (Costar 3603, Fisher 07-200-565). Do not use clear or white plates. Allow cells to attach and settle overnight.
  2. Wash cells with PBS 1X the next day.
  3. Add treatment media to each well using 100uL/well:
    1. Control :                        no treatment              (Biomedical Technologies BT-902)
    2. DiI AcLDL:                  10ug/mL DiI-AcLDL (Biomedical Technologies BT-906)
    3. AcLDL +DiI-AcLDL:  10ug/mL DiI-AcLDL + 200ug/mL AcLDL (unlabeled)

Note: Make sure you do not vortex these solutions, and that they are stored at 4C but not lower, since AcLDL will clump if frozen or if you place the vials on ice.

  1. Incubate cells for 4hours, 37C, in the dark.
  2. After incubation time, follow steps below to read fluorescence. Make sure you turn on fluorometer at least 30 minutes before you plan to use it, and that you have all materials ready.

Read fluorescence
1. Wash cells – After incubation, remove treatment media and wash cells with :
3x with 1xPBS
2. Add 50µL PBS to each well (96-well plates)
3. Read DiI fluorescence (no fixation):        Ex/Em           530/590
                       
Nuclear staining
1. Remove PBS using multi-channel pipet.
2. Fix cells – Add 100uL  4% Para-formaldehyde (PFA at 37˚C). Incubate RT for 10 minues.
3. Wash cells 1x with 100uL PBS, using multi-channel pipet.
4. Stain with nuclear dye: DAPI for the BMS plate (Poly-D-Lys coated) and Sytox for regular black plates
            DAPI              5,000x in PBS             100µL/well                  10 min
(3µM final; DAPI, Molecular Probes D-1306)
or
SytoxGreen     5,000x in PBS             100µL/well                  10min
(1µM final, 5mM stock)
5. Wash 3x with PBS—Remove staining solution and wash 2x with PBS, RT, using multi-channel pipet.
6. Add 50µL PBS to each well.
7. Read fluorescence of nuclear dye and DiI (fixed).
                        Excitation max                       Emission max
DiI                               530                                          590

DAPI                          358                                          461

SytoxGreen                485                                          530

Note: Read first in the SpectraMax plate reader, then in Jesus’ plate-reader.

 


Watson AD, Leitinger N, Navab M, Faull KF, Hörkkö S, Witztum JL, Palinski W, Schwenke D, Salomon RG, Sha W, Subbanagounder G, Fogelman AM, Berliner JA. Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo. J Biol Chem. 1997 May
23;272(21):13597-607. PubMed PMID: 9153208.

Bennett BJ, Farber CR, Orozco L, Kang HM, Ghazalpour A, Siemers N, Neubauer M, Neuhaus I, Yordanova R, Guan B, Truong A, Yang WP, He A, Kayne P, Gargalovic P, Kirchgessner T, Pan C, Castellani LW, Kostem E, Furlotte N, Drake TA, Eskin E, Lusis AJ. A high-resolution association mapping panel for the dissection of complex traits in mice. Genome Res. 2010 Feb;20(2):281-90. Epub 2010 Jan 6. PMID: 20054062.