Protocols
Below are the current protocols in use in the Blazar Lab.
ACK Lysis Buffer
ACK Lysis Buffer
For 1 L | |
0.15 M NH4Cl | 8.28 g NH4Cl |
1 mM KHCO3 | 1 g KHCO3 |
0.1 mM Na2EDTA | 37.2 mg Na2EDTA |
- Add 800 ml H2O
- Adjust pH to 7.2-7.4 (with 1 N HCl)
- Add H2O to 1 L
- Filter sterilize through a 0.2 mm filter
- Store at room temperature
Annexin V Staining for FACS
Annexin V Staining for FACS
- prepare 1 ml annexin V buffer/ tube (10x stock) in H2O
- do standard surface staining
- wash cells 1x with FACS buffer (PBS 2% FCS)
- wash cells 1x with annexin V binding buffer
- resuspend cells in 100 ml annexin V binding buffer
- add 3 ml annexin V
- optional: add 3 ml PI from kit
- vortex, incubate for 15 min at RT in the dark
- add 300 ml Annexin V buffer
- analyze by FACS immediately
CFSE Staining
CFSE Staining
- Set cells at 5 x 106/ml in PBS
- Add equal volume of 2 mM CFSE in PBS
- Stock conc.: 5 mM Þ 1 : 2500 dilution
- Incubate at RT for 2-3 min, shaking
- Stop reaction by adding FBS to a final concentration of 10%
- Wash 2x with PBS 2% FBS
In Vitro Suppression Assay
In Vitro Suppression Assay
- Use CD4+CD25- cells as responder cells
- Use CD4+CD25+ cells as suppressor cells
- Use irradiated syngeneic T cell depleted spleen cells as stimulators
- Set all cell populations at a concentration of 1.5 x106 cells/ml
- Mix responder : stimulators : suppressors 1 : 1 : 1
- Positive control: no suppressor cells, add equal volume medium instead
- Titration of suppressor cells: prepare dilutions of suppressor cells and add equal volumes to stimulators and responders
- add a CD3 (2C11 purified) at 0.5 m g/ml, mix well
- put 200 m l of cell suspension/well into 96 well plate (0.1 x106 cells of each cell population), at least 3 wells/condition
- incubate for 3 days at 37 ° C
- pulse plate with 3H-Thymidine
- harvest cells after 16-18 h
- dry filter and count
Mouse Tail DNA Prep
Mouse Tail DNA Prep
Stock solutions:
1M Tris-Hcl
MW 157.6 g/mol (15.76 g in 100 ml)
10 % SDS
10 g in 100 ml
50 mM EDTA
MW 372.24 g/mole (1.861 g in 100 ml)
1.5 M NaCl
MW 58.44 (8.766 g in 100 ml)
Lysis buffer:
for 100 ml | |
00 mM Tris-HCL pH8.5 | 10 ml 1M Tris-HCl |
0.2 % SDS | 2 ml 10 % SDS |
5mM EDTA | 10 ml 50 mM EDTA |
200mM NaCl | 13.3 ml 1.5 M NaCl |
add proteinase K (10-15 mg/ml) fresh each time
- Anaesthetize mouse (Isofluorin) and cut 1-1.5 cm tail into buffer tube. Treat wound with antibiotic
- Incubate sample in 55°C water bath over night. Sample should be clear; hair and bone on the bottom of the tube
- Invert tube several times to stir up DNA (or vortex). Centrifuge for 10 min at 14 000 rpm at room temperature.
- Prepare 1.5 ml tubes with 500 ml isopropanol and add supernatant. DNA precipitates. Shake vigorously.
- Prepare 1.5 ml tubes with 250 ml 8 mM NaOH
- Spool DNA using a pipet tip and put it into NaOH. Vortex until dissolved.
- Add 16.5 ml 0.1 M Hepes to neutralize pH. Vortex.
- Store DNA at –20°C
Spleen Stimulator Prep
Spleen Stimulator Prep
- Harvest spleen (into PBS/2% FCS in PBS), mesh, suspend and filter
- Lyse RBC with ACK lysis buffer (1-2 ml), wash 2-3x with PBS/2% FCS
- Resuspend and count
- Set to 20 x 106 cells/ml for mAb binding
- Add anti-Thy1.2 (30H12) and anti-NK1.1 (PK136) mAb at 20 ug/ml
- Incubate on ice for 30 min
- Add Nieffenegger Rabbit complement at 1:10 dilution
- Incubate at 37°C for 45 min, shake occasionally, wash 2x
- Resuspend in DMEM complete (DMEM, 10 % Hyclone characterized FCS, 5 % supplement, 100 mM 2-ME)
- Irradiate at 3000 Cs
- Count cells and set at the desired concentration
TRIzol RNA Isolation
TRIzol RNA Isolation
TRIzol reagent (GIBCOBRL/Life Technologies/Invitrogen Cat# 15596-026)
- Spin cells for 5 min at 5 000 rpm @ RT
- Add 1 ml TRIzol ( per 5-10 x 106 cells, resuspend cells
- Incubate for 5 min @ RT
Extraction
- Add 0.2 ml chloroform per 1 ml Trzol
- Shake tube vigorously by hand for 15 sec
- Incubate for 2-3 min @ RT
- Centrifuge for 15 min at 12 000 x g (12 000 rpm Spectrafuge 16M) at 4 ° C
- Transfer aqueous phase to a new tube
Precipitation
- Add 0.5 ml isopropanol (2-propanol) per 1 ml TRIzol
- Incubate for 10 min @ RT
- Spin for 10 min at 12 000 x g (12 000 rpm) @ 4 ° C
- Remove supernatant
- Add 1 ml per 1 ml TRIzol 75 % Ethanol
- Vortex
- Spin for 5 min at 7 500 x g (9 600 rpm) at 4 ° C
- Remove supernatant
- Air-dry pellet
- Resuspend RNA in Rase-free water
- Incubate for 10 min at 55-60 ° C
DNase I treatment
DNase I, Amplification grade (Invitrogen Cat# 18068-015)
- Reaction mix
- 1 mg RNA
- 1 ml 10x DNase reaction buffer
- 1 ml DNase I (1U/ml)
- add RNase-free water to 10 ml
- incubate for 15 min at room temperature (not longer!)
- add 1 m l of 25 mM EDTA to stop reaction
- incubate at 65 ° C for 10 min
- Store at –70 ° C
Yield determination
- Dilute sample in TE buffer (100 ml total)
- Measure OD260
- OD260/280 should be about 2.0
- 1 OD260 = 40 mg/ml