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

FAC-Sorting for RNA Isolation

FAC-Sorting for RNA Isolation