Preparation of Chk2 Inhibitor II stock solution
- Dissolve 1 mg of Chk2 Inhibitor II (EMD Millipore 220486) in 275 ul of DMSO to a final
concentration of 10 mM.
- Make 20 ul aliquots and store at -20°C. Stock solutions are stable for up to 6 months at
this temperature.
Thawing PBMCs
- Prepare 15 ml of thawing media and pre-warm at 37°C.
o 13.5 ml of IMDM/GlutaMAX
o 1.5 ml of FBS
o 30 ul of benzonase (= DNase I; add just before thawing the cells)
- Thaw one vial of PBMCs in the water bath and quickly transfer the contents to a tube
with 7.5 ml pre-warmed thawing media.
- Spin cells at 250 x g for 5 min at RT.
- Pour off the supernatant and resuspend the cells in 5 ml of thawing media.
- Count the cells (include trypan blue staining to estimate the percentage of live/dead
cells).
- Spin cells at 250 x g for 5 min at RT.
B cell isolation
- Prepare 5 ml of buffer (PBS with 2% FBS and 1 mM EDTA)
o 4.9 ml of PBS
o 100 ul of FBS
o 10 ul of 0.5 M EDTA
o 2.5 ul of 10 mM Chk2 Inhibitor II
- Resuspend the cells in buffer at a concentration of 50 million cells/ml and transfer to a 5
ml sterile filter-cap tube by pipetting the cell suspension through the filter.
- Add 50 ul Cocktail Enhancer (StemCell EasySep Human B cell Isolation Kit) for each 1 ml
of cell suspension and mix gently by swirling the tube.
- Add 50 ul Isolation Cocktail for each 1 ml of cell suspension and mix gently by swirling
the tube.
- Incubate for 5 min at RT
- Vortex the RapidSperes for 30 sec.
- Add 50 ul/ml RapidSperes to the cells and mix gently by swirling the tube.
- Add buffer to a total volume of 2.5 ml and mix gently by pipetting up and down 2-3
times.
- Place the tube without the cap into the magnet and incubate for 3 min at RT.
- Pour the enriched cell suspension into a new 5 ml tube. Leave the magnet and tube
inverted for 2-3 sec, but do not shake or blot off any drops that may remain hanging
from the mouth of the tube.
- Place the tube with the cell suspension on the magnet and incubate for 1 min at RT.
- Pour the enriched cell suspension into a new 5 ml tube.
Cell staining
- Prepare 20 ml of PBS with 1% BSA (from 35% BSA stock solution) and cool to 4°C.
o 19.4 ml of PBS
o 570 ul of 35% BSA
o 10 ul of 10 mM Chk2 Inhibitor II
- Transfer ~100k cells to each of four new 5 ml test tubes for compensation in flow
cytometry.
- Pellet the cells in all five tubes via centrifugation at 250 x g and 4°C for 5 min.
- Prepare a staining master mix in a microcentrifuge tube: 50 ul of staining volume per 25
million cells. See below for details on the composition of a master mix for one sample.
Antibody
CD20-PE-Cy7
CD27-APC
IgG-FITC
PBS-1% BSA (wt/vol)
-
-
-
Vendor
BioLegend 302311
BioLegend 302809
BD 560952
Volume (ul)
2.5
2.5
10
35
Resuspend the cell pellet from the original tube in 50 ul of the master mix.
Add PBS-1% BSA to each of the four compensation tubes (see table for amount). Add a
single antibody to three of the tubes as compensation tubes as shown in the table below.
One tube without antibody is used as an unstained control.
Tube ID
1
2
3
4
5
-
Clone
2H7
O323
G18-145
Antibody name
Master mix
CD20-PE-Cy7
CD27-APC
IgG-FITC
-
Antibody volume (ul)
50
2.5
2.5
10
-
PBS/BSA volume (ul)
0
47.5
47.5
40
50
Incubate the tubes at 4°C for 30 min in the dark (fridge).
Resuspend the cells with 1 ml of PBS-1% BSA using a P1000. Mix the suspension well and
add 2 ml more of PBS-1% BSA.
Pellet the cells by centrifuging the tubes at 250 x g and 4°C for 5 min and remove the
supernatant.
Resuspend the compensation controls and the unstained control in 300 ul of PBS-1% BSA.
Resuspend the samples in PBS-1% BSA at a concentration of 20-30 million cells/ml
(minimum volume = 300 ul). Transfer the resulting suspensions to 5 ml sterile filter-cap
tubes.
Store the suspensions at 4°C and cover them with foil until ready to proceed to cell sorting
(recommended within 3 h).
Keep on ice when transferring samples to the flow cytometry core and prior to
analysis/sorting.
27JAN17:
- 27.5 million cells after thawing