Preparation:

PMA   -use at 10ng/ml final concentration. Stock at 1mg/ml in enthol or DMSO, aliquots

Ionomycin       -use at 500ng/ml final concentration. Stock at 1mg/ml in enthol or DMSO,

Anti-CD3e      -use at 5ug/ml final concentration. Stock from R&D systems or Pharmingen.

Anti-CD28      -use at 5ug/ml final concentration. Stock from R&D systems or Pharmingen.

Brefeldin A (BFA)        -use at 10ug/ml final concentration. Dissolve BFA at 10mg/ml in ethanol, then the Stock from R&D systems or Pharmingen.

4%PFA in PBS Need to dissolve at 60degree, aliquot and store at –80degree.

PermWash      PBS containing 0.1% saponin, 0.5%BSA, and 0.1%Azide.

FACs buffer    PBS containing 0.5%BSA, and 0.1%Azide.

Procedures

1. Surface staining and wash; Discard the FACS buffer,

For PMA+Ca++-stimulated T cells, CD4 staining can be done with the cytokine staining step since PMA downregulate CD4 from the surface.

2. Put 75ul(for 1million cells) 4%PFA in PBS, vortex briefly, then 20-30 minutes at RT,

3. Alternatively, add 1ml FACS buffer and continue staining in second day.

4. Wash with 1ml PermWash,

5. Repeat Step d.

6. Put 50ul of PermWash with Fcblocker,

7. RT, 10 min,

8. Add 50ul PermWash with antibodies against cytokines.

9. 4 degree for 20-30 minutes.

10. Wash with 2ml PermWash,

11. Discard the supernatant.

12. Put FACS buffer and ready for FACS. (The cell pellet can kept for at least for 2days at 4 degree till ready for FACS)

Propidium Iodide for DNA Content

Note: After running samples with Propidium Iodide the cytometer needs thorough cleaning with 10% bleach and distilled water!

Solutions

·       70% ethanol

·       ribonuclease (100 µg/ml DNase free, Sigma)

·       propidium iodide ( 50 µg/ml in PBS)

Procedure

1.     Harvest cells. Spin at 1200 rpm for 5 minutes.

2.     Discard supernatant; add 1 ml ' fridge cold' 70% ethanol to 1x106 cells while vortexing the cell pellet gently.

3.     Fix for at least 30 min. at 4° C.

4.     Spin at 2000 rpm for 5 min. Once in 70% ethanol, samples may be kept for up to 2 weeks.

5.     Discard supernatant and resuspend cells in1 ml PBS. Spin 2000 rpm for 5 min.

6.     Repeat step 5 twice more.

7.     Add 100 µl ribonuclease (100 µg/ml, DNase free, Sigma). Leave at room temperature for 15-30 min.

8.     Add 400 µl propidium iodide (50 µg/ml in PBS).

Note: PI is potentially mutagenic so wear gloves.

Before running the samples stained for DNA content the cytometer's linearity, resolution and doublet discrimination capability should be verified. DNA QC kit is necessary and should be ordered from Becton Dickinson (Cat. No. 349523).

The kit is composed of :

CEN- chicken erythrocyte nuclei, used in setting cytometer's photomultiplier tube (PMT) voltages and amplifier gains, and providing information regarding instrument linearity and resolution;

CTN - calf thymocyte nuclei, a cycling cell component that allows assessment of proper function of doublet discrimination module (DDM) or pulse processing. Please follow the instructions included in the kit for quality control and set-up of the machine.

9.     Acquire data on a FACScan or FACSCalibur, the cytometer should be triggered on the PI signal. Primary gate is on forward scatter (FSC) against right angle light scatter (SSC).

10.  A secondary gate should be placed around the single cell population on a pulse area versus pulse width dot plot.

The measurement of cell cycle parameters can be analyzed by modeling programs which fit the best gaussian distribution curve to each peak, Go-G1, G2-M and then calculate the resulting S-phase. Modfit software from Verity Software House (Verity@vsh.com) can be used.

Calcium flux detection:

- Indo-1-AM: 1mM = 1mg/ml (1000x, endconc. 1ug/ml)

  (Mol. Probes/invitrogen, cat. # I1226)

- HEL: 10mg/ml stock in water (SIGMA, cat. # L6876)

- Anti-IgM: F(ab’)2 fragment goat anti-mouse IgM) 1.3 mg/ml

  (Jackson Immuno cat # 115-006-075)

- CLM (cell loading medium): RPMI-1640, 5%FCS, pen/strep

• Isolate splenocytes from HEL Tg or use cell line (Bal17 or other)

• Spin down, do RBC lysis (in case of splenocytes)

• Wash cells and count

• take desired amount of cells and spin down

• Resuspend cells to 1 x 107 /ml in CLM

• add indo-1/AM to endconc. of 1 ug/ml (=1uM) and incubate for 30 min at 37C in dark (shake a few times for even loading)

If you want to do a staining on the cells:

• Wash and spin down, resuspend again to 1x107/ml

• stain for 15 min at RT in dark

• wash cells

• Dilute cells to 1x106 cells per ml (you can use a bit more concentrated if desired but 1x106 cells/ml is enough for a cell line)

• add 1ml per facs tube

• Analyze The ratio of indo-1 violet/blue using UV laser (I use Vantoo machine with the DIVA software)

• Collect data for 30-50 sec to establish baseline violet/blue ratios (bound ~420nm, free ~510nM)

• Stimulate cells with HEL Ag, or anti-IgM F(ab’)2 (or p.c. Ionomycin) and collect data for a total of 5-6 min.

• Range of HEL ag I use: effects on splenocytes of HEL tg start at +/- 10ng/ml but you can use much more depending on purpose (i.e 250ng/ml)

• Range of anti-IgM I use: starting at 0.5 ug/ml up to 5 ug/ml is optimal range for BAL17 cells

• You can use Ionomycin as a positive control (all cells should respond)

·        Data is analyzed afterwards using flowjo software

DNA Isolation From BAC & PAC Clones  (BACPAC Resources)

 This is a rapid alkaline lysis miniprep method for isolating DNA from large BAC (pBACe3.6) or PAC clones.   It is a modification of a

standard Qiagen-Tip method that uses no organic extractions or columns.  The method works very well for doing

analytical restriction digests of PAC clones and can be scaled up if necessary.

1. Solutions

         P1 (filter sterilized, 4oC)

            15mM Tris, pH 8

            10 mM EDTA

            100 ug/ml RNase A

        P2 (filter sterilized, room temp)

            0.2N NaOH

            1% SDS

        P3 (autoclaved, 4oC)

            3M KOAc, pH 5.5

2. Method

 1. Using a sterile toothpick, inoculate a single isolated bacterial colony into 2 ml TB (or LB)media supplemented with 25 ug/ml kanamycin (for PACs) or 20 ug/ml chloramphenicol (for BACs). Use a 12-15 ml snap-cap polypropylene tube. Grow overnight (up to 16 h) shaking at 225-300 rpm at 37°C.

 2. Remove toothpicks using forceps. Centrifuge (SM24 or similar rotor) at 3,000 rpm for 10 min. of spin in the Sorvall. The temperature ot the spin is not critical at this stage.

 3. Discard supernatants. Resuspend (vortex) each pellet in 0.3 ml P1 solution. Add 0.3 ml of P2 solution and gently shake tube to mix the contents. Let sit at room temperature for 5 min or so. The appearance of the suspension should change from very turbid to almost translucent.

 4. Slowly add 0.3 ml P3 solution to each tube and gently shake during addition. A thick white precipitate of protein and E. coli DNA will form. After adding P3 solution to every tube, place the tubes on ice for at least 5 min.

 5. Place tubes in the SM24 rotor and spin at 10,000 rpm for 10 min at 4 oC.

 6. Remove tubes from centrifuge and place on ice. Transfer supernatant using a P1000 or a disposable pipette to a 1.5 ml eppendorf tube that contains 0.8 ml ice-cold isopropanol. Try to avoid any white precipitate material. Mix by inverting tube a few times; place tubes on ice for at least 5 min. 

At this stage, samples can be left at -20° C overnight.

 7. Spin in cold microfuge for 15 min.

 8. Remove supernatant and add 0.5 ml of 70% EtOH to each tube. Invert tubes several times to wash the DNA pellets. Spin in cold microfuge for 5 min.

Optional --repeat step 8.

 9. Remove as much of the supernatant as possible. Occasionally, pellets will become dislodged from the tube so it is better to carefully aspirate off the supernatant rather than pour it off.

 10. Air dry pellets at room temp. When the DNA pellets turn from while to translucent in appearance, i.e., when most of the ethanol has evaporated, resuspend each in 40 ul TE.  Do not use a narrow bore pipets tip to mechanically resuspend DNA sample; rather, allow the solution to sit in the tube with occasional tapping of the bottom of the tube. For large PAC clones resuspension may take over 1 hour.

Overview

CFSE is used to fluorescently label live cells in order to track them. 5,6-CFDA/SE is membrane permeable, but trapped in the cell when converted by intracellular esterases. CFSE is equally partitioned to daughter cells during division and can be used to measure cell proliferation.

Materials

• 15 mL polypropylene tubes

• PBS

• 5,6-CFDA/SE Invitrogen C1157 dissolved in DMSO at 5 mM and stored at -20°C.

Procedure

1. Suspend PBMCs at 10x106 cells/mL in PBS alone.

2. Ensure that cells are uniformly suspended when CFSE is added.

3. If CFSE negative control is needed, remove cells now.

4. Make '2X' concentration (10 μM) in PBS

   • For example: add 5 mL PBS + 10 μL 5 mM CFSE

5. Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.

6. Place in 37°C H2O bath x 10 min.

7. Wash in 10 mL complete medium.

8. Resuspend in medium at desired densit

Colony PCR

Colony PCR

 1. Master mix (for 96 well plate):

 H2O                                        7.6 ml

Tag                                          200 ul

PCR buffer(10X)                      1    ml

dNTPs (10X)                           1    ml

Primers  5' (100uM)           100ul

             3' (100uM)      100ul

aliquot into 96-well plate at 50 ul/tube

2. Fresh grown colonies (or 5ul our of 2ml of fresh grown bacterial culture) into 100 ul H2O, Heat at 100 C, 5’ (or boil 5’)

3. 5 ul of heated colony suspension into PCR tube containing 25 ul rxn mix.

4.  PCR

    95 C            5 min

    94 C                        30``

    55 C                        30``

    72 C                    1` (for products between 200-1kb)

            30 X

 5. Loading 1% metaphor agrose gel with DNA ladders.  

Preparation:

1. capture antibody:

    resuspend in 1 ml PBS ---720ug/ml (180X)

    aliquots and store in –80 C

    working concentration: 4.0 ug/ml in PBS

2. detection antibody:

   resuspend in 1.0ml PBS---72ug/ml (180X)

   aliquots and store in –80 C

   working concentration: 0.4ug/ml in PBS 

3. standards

IL-4

3. • reconstitute in 0.5ml of Reagent Diluent, ---

      100ng/ml (100x of highest dilution)

3. • aliquots and store in –80 C

   • working standard curve:  7 point 2-fold serial dilution 

                  1000pg/ml,    500pg/ml,      250pg/ml,      125mg/ml,

62.5pg/ml,     31.25pg/ml,    15.625pg/ml,    0 

IFN-gamma

3. • reconstitute in 0.3ml of Reagent Diluent, ---

      100ng/ml (50x of highest dilution)

3. • aliquots and store in –80 C

   • working standard curve:  7 point 2-fold serial dilution 

                  2000pg/ml,   1000pg/ml,      500pg/ml,      250mg/ml,

125pg/ml,     62.5pg/ml,    31.25/ml,    0  

4. wash buffer

0.05% Tween-20 in PBS 

5. reagent diluent

1%BSA in PBS, 0.2uM filtered 

6. substrate solution

1:1 x mixture of color reagent A and B  right before use 

7. stop solution

2N H2SO4 

Assay

    Day 1

1.      Plate Preparation

A.     dilute  the capture antibody to the working concentration in PBS w/o carrier protein.

B.     Immediately coat 96-well microplate with 100ul/well of the diluted capture antibody

C.     Seal the plate and incubate overnight at room temperature.   

    Day 2

1.      wash plate:

aspirate each well and wash with 400ul /well of wash buffer 3X,  total. 

2.      block plate

adding 300ul of reagent diluent /well

incubate at RT for >= 1 hour 

3.      wash blocked plate

aspirate each well and wash with 400ul /well of wash buffer 3X,  total. 

4.      Assay

Standard curve: 7 point 2 fold serial                   x2 (duplicates)

100ul of sample, with 5 fold serial dilution,         x3 (triplicates)

RT, 2 hours incubation 

5.      aspirate each well and wash with 400ul /well of wash buffer 3X,  total. 

6.      Add 100ul of Detection antibody, diluted in Reagent Diluent. Incubate for 2 hours at room temperature. 

7.      aspirate each well and wash with 400ul /well of wash buffer 3X,  total. 

8.      100 ul/well of working dilution of streptavidin-HRP

RT, 20 minutes, avoid light 

9.      aspirate each well and wash with 400ul /well of wash buffer 3X,  total. 

10.  100 ul/well of Substrate Solution mixture

RT, 20 minutes, avoid light 

11.   50 ul of stop solution to each, mix by tapping 

12.  microplate reading at 450nm, subtract reading at 540 or 570 as wavelength correction.