Huang Lab: Resources and Services

Immunization Protocols:

(Updated 7/20/06)

Interperitoneal (i.p.) Injection
Intranasal Challenge (i.n.)
Immunization Schedule
General FACS Staining Protocol
FACS Staining Buffer
PI Solution
Protocol for Stable Small Interfering RNA (SiRNA)
Lipofectamine 2000 Transfection of Phoenix Cells

 
 

Interperitoneal (i.p.) Injection

  1. Chicken OVA Albumin (Sigma #) is stored at 4°C in Room 5654 in a plastic desiccant can.
  2. Obtain, a 15 mL conical tube, a piece of weigh paper, and little spoon and walked down to the scale.
  3. Measure out 5 mg of the OVA. (On this scale it reads 0.005g) Pour the OVA into the 15 mL conical tube. Try to make sure all of the OVA is at the bottom of the tube.
  4. Add 5 mL of commercial 1x PBS. The concentration will be 1ug/ul.
  5. Vortex the tube a few times.
  6. Sterile filter through a 0.45 um filter into a new 15 mL conical tube.
    *** Always make OVA solution fresh the day of the injection.***
  7. First obtain a 100 mL Flask and a small stir bar. Place the flask of the stir plate on an angle and start the stir bar
  8. Mix the OVA and Alum in a 1:1 ratio
  9. Add the OVA first, then add Alum (Pierce: Imject Alum, Cat # 77161) dropwise and stir for 30 minutes or longer.
  10. Take the mixture into a 1 cc syringe and inject 0.2 mL (containing 100 ug OVA) into mouse peritoneal cavity.  ***Mice are ALWAYS numbered by toe clip.*** Make sure the toe clip number and the litter number is correct before injection. When injecting, make sure to avoid injecting into intestines by inserting the needle into the lower part of abdomen (near groin). Stop insertion once you feel the needle has pierced through the abdomen wall, the resistance is dramatically reduced once the needle is through the abdominal wall.

 

Intranasal Challenge (i.n.)

  1. Prepare fresh OVA by weighing the Chicken OVA Albumin. Weight out 10 mg (0.010g) on the scale in Dr. Witte’s lab.1. 
  2. Add 5 mL of sterile 1x commercial PBS and sterile filter with 0.45um filter.
  3. Anesthetize mice with half a bottle top of Halofane absorbed in a cotton ball, placed in the jar. Leave the cover of the jar open and carefully observe the mouse breathing. 
    ***This process requires experience.***
  4. Pipet 100 l of OVA into the nose of anesthetized mice (50 ul into each).
    Often mice choke, CPR is often required to revive the mice. 
    ***This process requires experience.***

 

Immunization Schedule

  • Mice are injected (i.p.) on day 0 and day 14.
  • 7 days later mice will be challenged with daily intranasal injections for three consecutive days.
  • 48 hours after last i.n. mice will be sacked.
  • Tissue samples will be taken for FACS analysis.
  • Blood will be collected for serum IgE analysis.
  • Sections of Lung will be collected for Histology.

 

General FACS Staining Protocol

Huang Lab protocol
Last update 3/16/06

  1. Resuspend cells (1x 105- 106) in 200 ml (l) of FAC buffer in a FACS staining tube.
  2. Add 2l of anti-FcRiii (2.4G2, 0.5 mg/l) and incubate on ice for 5 minutes.
  3. Add 1l of antibody of choice for 20-60 minutes.
  4. Wash 1x in 2 mL of FACS Buffer. (0.5 ~ 1 mL in small FACS tubes).
  5. Resuspend in 200l of FACS Buffer (100l in small FACS tubes) and add PI solution (2l/1 mL of FACS buffer).
  6. Collect cells and store data by FACS using Huang 4 C setting.

 

FACS Staining Buffer

500mL:
  • FBS 10 mL (working 2%)
  • 10% Sodium Azide 0. 5 mL (working 0.01%)
  • 1x PBS 485 mL

 

PI Solution

  • Stock concentration is 100 ug/ml.
  • Dilute stock to working concentration of 1 ug/ml. 

 

Protocol for stable small interfering RNA (SiRNA)

Huang Lab, last updated 12/9/04

 

Design:

For example, to construct IRS2 SiRNA, we will first choose several sequences that may, or have been shown, to function as small interfering RNA from SiRNA databases. 

A hairpin of the selected sequence will be constructed by arranging the selected sequence in sense and antisense direction and an insert of loop sequence of eight nucleotides (UAGCUUCG). Expression of the hairpin structure will be under the control of human RNA polymerase III promoter. 

For the control, we use a random sequence:
5’-CGAACGAGUACCGUACACUUAGCUUCGAGUGUACGGUACUCGUUCGUU-3’

The hairpin and human polymerase III promoter will be synthesized and cloned into Banshee retroviral vector (gift of Dr. John Rossi, Beckman Research Institute of the City of Hope, CA). 

The effectiveness of SiRNA will be first tested on 32D cell line that express high level of IRS1/2.

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Lipofectamine 2000 Transfection of Phoenix Cells

Written by Yong, Huang lab, Last update: 10/12/06
(Modified from Dr. Jennifer’s protocol)

  • - Day 4
  1. Thaw Phoenix E cells, seed the cells in 10 ml cDMEM in 10 cm dish.
  • - Day 2
  1. Split the cells 1 to 5.
  2. Remove old medium completely and wash with PBS.
  3. Add Trypsin and remove it immediately.
  4. After beating the dish 2-3 times the cells will begin to move.
  5. Add Medium with FBS.
  • Day 0 (one flask yields roughly 10-15 X 106 cells)
  1. Coat dishes with l0 ug/ml poly D lysine (Sigma P0899) for 5 minutes RT in H20. Stock is at l0 mg/ml, so use lul of stock/lml in one 6wpw.
  2. Wash wells 2X with PBS. Dry for 2 hours in the hood(it worked also without drying).
  3. Plate phoenix cells at 600,000/ml in 2ml in 6wpw in DMEM (10% FBS, but no antibiotics).
  4. Incubate overnight.
  • Day 1
  1. Dilute DNA (4 ug of plasmid of choice plus 1.5ug of pCLECo) into 250ul Opti-MEM, VORTEX. Prewarm DMEM(without antibiotics and FBS).
  2. Dilute 10ul of lipofectamine (Life Technologies/Gibco) into 250ul of plain Opti-MEM, Pipet up and down 10X (Mix gently). Incubate 5 minutes at room temperature.
  3. Add lipofectamine forcefully to DNA and pipette 10X.
  4. Incubate for 20 minutes at RT (do not move this during that time).
  5. Remove the old cDMEM and wash twice with prewarmed PBS and add new prewarmed DMEM w/o antibiotics and FBS.
  6. Add the solution from the step 4 (0.5 ml) drop wise to cells, covering entire surface area. Move the plate to 4 directions: forward, back, right and left.
  7. Incubate for 4-6 h, add 0.3 ml pre-warmed FBS. 
  • Day 2
  1. Replace media with 4 ml pre-warmed DMEM (10%FBS, no antibiotics) if is it yellow.
  2. Change half medium if it is not so yellow.
  3. Move to a 32 degree incubator, incubate overnight.
  • Day 3
  1. Collect viral supernatant (24h viral supernatant).
  2. Spin to pellet down any cells or debris or filter with 0.45um filter. Ready to use.
  3. Replace media if you are going to collect viral supernatant at 48h.
  • Day 4
  1. Collect media again, spin or filter.
Note:
  1. Transfection efficiencies depend on both the fitness of the Phoenix cell culture and purity of the DNA. It is important to keep the virus-package cell line in good shape. Usually we package cells 1 to 4 or 1 to 5 every other day depending on the starting cell numbers. So cell density never exceeds 70% confluent. We also have tested the capacity of these cells after thawing to make cytokines. So we only use Phoenix E cell line two passages after thawing. Never use the cells after day 10.
  2. When changing the medium, make sure to pre-warm the medium. Otherwise cells will undergo stress and form clumps and never become flat.
  3. Cell density needs to be empirically determined since cell counting varies from person to person. Although cell counting is very important for seeding cells, we cannot pipette cells forcefully and separate cells completely because it will do great harm to cells.
  4. Sometimes the plasmid amount used for transfection needs to be optimized for maximal transfection rate. If needed, do a titration.
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