1.  Lin- cells are resuspended with 48-72hr retroviral medium and seeded in 6 well plate.

 2.  4-10ug/ml polybrene was added and cells were spin infected at 37 for 2 hours at 2000Xrpm.

 3.  Remove half retroviral medium and add 1/2 total volume of fresh medium with 10% FBS,  50uM of beta-ME, 20ng/ml SCF, 30ng/ml Flt3L, 10ng/ml of Il-6 culture o/n

 4.  Spin and remove medium and replace with 72-96hr retroviral medium with polybrene for another 1-2hrs at 2000 rpm.

 5.  Remove supernatant and resuspend cells in Fresh IMDM with 2% FBS at concentration of 1X10*6/100ul for injection. 100ul /mouse.

Materials:

1.    IMDM +10%FBS

2.    PBS +2% FBS

3.    L929 cell supernatant or GM-CSF

4.    70um cell strainer

5.    21g or 23g needles

6.    surgery tools

7.    NH4Cl solution (Stemcell Laboratory)

BMDM growth medium:

       IMDM+10%FBS+15%L929 SN (or 10ng/ml GM-CSF)

Protocol:

1.    Isolate femur and tabular bone from B6 mice, rinse off hairs before cutting open the bone

2.    Use 23g or 21g needle on 10ml syringe filled with cold PBS+2% FBS (3-5ml for one mouse) to flush marrow from the bone

3.    Pass marrow through 23g or 21g needles 4-6 times to separate cells

4.    Pass cell through a 70um cell strainer to remove cell clumps, bone, hair or other tissue

5.    Add 3 vol (~15 ml) of NH4Cl solution, incubate on ice for 10 min to remove red blood cells

6.    Spin down, 2000rpm (or 500 x g) for 5 min

7.    Resuspend cell pellet with cold PBS+2% FBS (20-50ml, depending on the cell quantity).  Count the cells.

8.    Spin down, 2000rpm (or 500 x g) for 5min.

9.    Resuspend cells at 1-1.5x10^6/ml with BMDM growth medium: IMDM + 10% FBS + 15% filtered L-929 medium.

10. Seed cells at 1-1.5x10^6/ml in 12 well plates.  15ml per 10cm plate. (Or, Keaton: one mouse for 50ml culture).  

11. Change fresh growth medium on day 3.

12. BMDM can be used for further study on day 7.

Remove BMDM from dish (it is better to use petri dish for BMDM culture)

DPBS(free Mg,Ca)/EDTA (0.5M) COLD

1.   Wash BMDM with PBS (cold)  after removal of medium

2.   Add DPBS(Ca-, Mg-) + 5mM EDTA (cold)

3.   Place cells on ice for 20-30 minutes (check every 10-15 min), tap the plate

Pipetting cells to collect!  These are good for FACS analysis

1) Coat a 24 well plate with 1 ml 5 mg/ml anti-CD3 antibody and 5 mg/ml anti-CD28 antibody containing PBS per well over night at 4-degrees.  Remove liquid from wells immediately prior to use. 

2) To each well add 106 naïve CD4 T-cells in 1 ml T cell medium  (10% FCS, Pen/Strep, L-Glu, 2-ME RPMI.)  For Th1 polarization also add 5 ng/ml IL-12 and 10 mg/ml anti-IL-4 antibody.  For Th2 Polarization add 25 ng/ml IL-4 and 10 mg/ml anti-IFN-gamma antibody.  Incubate at 37-degrees. 

3) After 24 hours of stimulation add IL-2 to a final concentration of 40 U/ml. 

4) After 72 hours of stimulation, remove the cells from stimulation, split them 1:3, and maintain for another 72 hours in the presence of IL-2 plus polarizing cytokines.  At this point analysis can be performed.

Isolation of thioglycolate-elicited peritoneal cells by peritoneal lavage

Materials:

Instrument tray                                    Set of instruments (Forceps/scissors)

Trash bag                                            Ice bucket/ice

70% EtOH squeeze bottle                   10 ml syringes containing collecting medium

Hemostats                                           50-ml conical tubes (4)

Tube tray                                             Small transfer pipets

1 ml syringe                                         3 ml syringes                                      

Paper towels                                       Latex gloves  

Respirator                                           Tape   

Lab coat                                              Marker and pen                      

Sleep Away (pentobarbital sodium) in 1 ml syringe  

3% thioglycolate broth (Sigma) (autoclaved) 

Collecting medium: 10 U/ml heparin/5% FBS in RPMI complete-18 ml/animal (keep ON ICE)

Cell wash medium: 5% FBS in PBS (keep ON ICE)

ACK-lysing buffer:  0.15 M NH4Cl, 1 mM KHCO3 and 0.1mM Na2 EDTA, pH 7.2-7.4

RPMI complete                        

        RPMI 1640 HEPES (Gibco) + 10% FBS (Atlanta Biol.)                

        100 U/ml penicillin

        100 mg/ml streptomycin

        2 mM L-glutamine

Protocol:

1.    Inject 2ml of 3% thioglycolate solution into the peritoneal space of the animal 3 days prior to the lavage procedure.

2.     3 days after thioglycolate injection, euthanized the animals injecting 150-200 mg/kg of Sleepaway into the hind limb (intramuscularly).

3.     After the heartbeat disappears (determined by palpating over the rib cage), take the animal and lay it on its back on 2-3 layers of paper towels.

4.     Apply 70% EtOH to the chest/abdominal region.

5.     Take the first set of sterile instruments and remove the skin from the ventral surface from the inguinal region to the trachea.

6.     Inject 6ml of collecting medium into the peritoneal cavity with a 10 ml syringe and massage the abdomen.

7.     Make a circular incision into the peritoneal layer on the mid-line (about 1cm in diameter)

8.     Insert a transfer pipette through the hole in the peritoneum and clamp the peritoneal surface using a hemostat to form a tight seal. 

9.     Extract the peritoneal wash fluid and place it into a 50ml tube. Keep on ice.

10.  Repeat injection of medium, massage and collection twice using fresh collecting media.

11.  Centrifuge the tubes for 10 min at 1260 rpm at 4°C and discard the supernatant.

12.  Wash the cells in 10 ml wash medium, centrifuge for 10 min at 1260 rpm at 4°C

13.  Add 10 ml of ACK lysing buffer to remove the contaminating erythrocytes. Repeat if necessary to eliminate all red blood cells.

14.  Wash the cells twice in 10ml wash medium, centrifuge for 10 min at 1260 rpm at 4°C.

15.  Re-suspend the pellet in 1ml of complete media and determine cell count and viabilityby trypan blue exclusion.

16.  Adjust volume for the desired cell/ml concentration and plate.

Modified by D.Bonilla from the guinea pig protocol (Laboratory Manual), according to Yamamoto T, 2007 and Saiki I, 1982.

References

Yamamoto, T. et al. Mycobacterium bovis BCG vaccination modulates TNF-alpha production after pulmonary challenge with virulent Mycobacterium tuberculosis in guinea pigs. Tuberculosis 87, 155-65 (2007).

Saiki, I. et al. Adjuvant activity of purified peptidoglycan of Listeria monocytogenes in mice and guinea pigs. Infect Immun. Oct;38(1):58-65 (1982).

Hepatocyte (Hc) and non-parenchymal cells (NPC) isolation protocol

From: Juli Bai, Feng Liu (UTHSA), Nov 2019

1.      Anesthetize the mice, using Ketamine/Xylazine. (Ketamine/Xylazine conc. used was 80mg/Kg & 10mg/Kg respectively.)

2.      Prepare the animal for surgery wiping the abdomen with ethanol, spread the limbs, and secure them to the dissection board.

3.      Dissect the peritoneal cavity and push the intestines to the right side using a cotton-tip; identify both the inferior vena cava and hepatic portal vein.

4.      Make the liver perfusion throughout inferior vena cava. Insert the needle through the vena cava and hold the needle in place with a clamp in order to secure it.

5.  Cut the hepatic portal vein.

6.      Pass 30 mL EGTA buffer (pH = 7.4) (37°C preheated) slowly.

7.      Pass 6 – 9 mL/mouse Collagenase type 2 (0.025g in 50 mL, 37°C preheated). Collagenase II final conc.: 0.5 mg/mL.

8.      When the liver is sufficiently digested (be soft), cut off the liver (make sure the liver is intact at this point) and transfer it to 1X HBSS/HEPES (with Ca2+ and Mg2+) buffer in a Petri dish while working on ice.

9.      Remove the liver capsule by cutting the liver lobes and continue until all of the big clumps are gone.

10.  Filter through 100-µm strainer.

11.  Spin 50g x 2 min at 4°C.

12.   Separate the supernatant (NPC) and Pellet (HC). Collect the supernatant (NPC) into a coated 50 mL tube (Tubes were coated with BSA to reduce sticking of Kupffer Cells to the tube), put NPC on ice & re-suspend the pellet (HC) with 1X PBS + 2% FBS.

13.   Wash the HC with 1X PBS + 2% FBS twice by repeated centrifugation and re-suspension (50g x 2 min at 4°C),

14.   Similarly, collect all supernatants which contain NPC from same mouse in tube and then centrifuge at 50g for 2min to discard the pellet (the rest of hepatocyte), and then centrifuge at 600g for 10min to get NPC pellet,

15.     Re-suspend NPC in 1X RBC lysis buffer, put on ice for 6-10 min, dilute with 1X PBS.

16.   Centrifuge at 600 g x 5 min, resuspend in 1X PBS with 2% FBS.

17.  If the viability is low, Percoll density separation step can be used to remove dead cells or cell debris.