Jenkins MHCII pulldown › Jenkins_Lab_2 › assets › pdf...Jenkins lab 3 8/4/08 To calculate the...
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Jenkins lab 8/4/08 1 Jenkins Lab MHCII Tetramer Pulldown Protocol - 8/4/08 check for updates at www.jenkinslab.umn.edu 1. Harvest spleen and all lymph nodes (inguinal, axillary, brachial, submandibular, cervical, mesenteric, para-aortic) from mouse. Mash over nylon mesh in 1-2 ml ice-cold complete EHAA and filter through nylon mesh into a 15 ml polypropylene conical centrifuge tube. Rinse mesh with another 2 ml. Top off with cold sorter buffer (PBS + 2% FBS, 0.1% NaN 3 ) and spin down (Beckman tabletop centrifuge 1600 rpm 5 min 4C). 2. Aspirate and add Fc block (2.4G2 SFM supe + 2% mouse serum, 2% rat serum, 0.1% NaN 3 ) to a final volume about twice that of the pellet itself (use graduations on tube to estimate volumes). Be sure to remove all droplets of media from the sides of the tube. Resuspend pellet by rigorous vortexing. The pellet size for a naive mouse is about 100 µl, in which case the final volume should be 200µl. For immunized mice, you may need to increase the final volume. Remember to leave room for the volume of tetramer to be added. 3. Add tetramer to the sample at a final concentration of 5-25 nM, depending on the dose response curve for the current production lot of tetramer. Generally, the optimal concentration is 10 nM. Concentrations above 25 nM may lead to increased background staining. Vortex and incubate in a room temperature water bath in the dark for exactly 1 hour. 4. Wash with 15 ml cold SB. The cells should be kept on ice from now on. 5. Add SB to the pellet to a final volume of 200 µl. Vortex to resuspend. Add 50 µl Miltenyi anti-PE microbeads. Vortex again and incubate on ice in the dark for 30 min. 6. Wash with 15 ml SB. 7. While spinning, set up a Miltenyi LS column on a MidiMACS magnet and pre-rinse with 3 ml SB (discard flow-through). Place a new 15 ml conical tube under column. 8. Resuspend pellet in 3 ml SB and apply to the column, filtering through nylon mesh in the process. 9. Rinse the original sample tube with 3 ml SB and apply to the column, also rinsing the nylon mesh in the process. Discard nylon mesh. Add two more washes of 3 ml each to the column for a total of three x 3 ml washes. Continue collecting the flow-through into the same unbound collection tube. 10. Remove the column from the magnet and place on top of a new 15 ml conical tube. Add 5 ml SB and force the buffer through in one motion with the plunger. Discard column and plunger. 11. Spin down the bound and unbound fractions. Carefully aspirate all but about 40 µl of buffer from the bound fraction tube. Resuspend the pellet to exactly 95 µl with Fc block. Aspirate the unbound fraction tube and resuspend to 2.0 ml with Fc block. For each fraction, take out 5 µl and mix with 200 µl counting beads (Caltag-Invitrogen #PCB-100) in a 5 ml FACS tube. 12. Stain and analyze by flow cytometry immediately. For the bound fraction, add antibody cocktail directly to the remaining ~90 µl cells in the 15 ml conical tube. For the unbound fraction, remove 90 µl and stain with antibody cocktail in a 5 ml FACS tube. Incubate on ice for 30 min. Wash samples with 5 ml SB. For the bound fraction, aspirate the supernatant, resuspend the pellet in 250 µl SB, and transfer to a 1.2 ml FACS microtube (fits inside a regular 5 ml FACS tube). Rinse the 15 ml tube with another 250 µl and pool together in the same microtube. For the unbound fraction, decant the supernatant and resuspend the pellet in 2.0 ml SB.
Transcript of Jenkins MHCII pulldown › Jenkins_Lab_2 › assets › pdf...Jenkins lab 3 8/4/08 To calculate the...
Jenkins lab 8/4/08 1
Jenkins Lab MHCII Tetramer Pulldown Protocol - 8/4/08 check for updates at www.jenkinslab.umn.edu
1. Harvest spleen and all lymph nodes (inguinal, axillary, brachial, submandibular, cervical,
mesenteric, para-aortic) from mouse. Mash over nylon mesh in 1-2 ml ice-cold complete EHAA and filter through nylon mesh into a 15 ml polypropylene conical centrifuge tube. Rinse mesh with another 2 ml. Top off with cold sorter buffer (PBS + 2% FBS, 0.1% NaN3) and spin down (Beckman tabletop centrifuge 1600 rpm 5 min 4C).
2. Aspirate and add Fc block (2.4G2 SFM supe + 2% mouse serum, 2% rat serum, 0.1% NaN3) to a final volume about twice that of the pellet itself (use graduations on tube to estimate volumes). Be sure to remove all droplets of media from the sides of the tube. Resuspend pellet by rigorous vortexing. The pellet size for a naive mouse is about 100 µl, in which case the final volume should be 200µl. For immunized mice, you may need to increase the final volume. Remember to leave room for the volume of tetramer to be added.
3. Add tetramer to the sample at a final concentration of 5-25 nM, depending on the dose response curve for the current production lot of tetramer. Generally, the optimal concentration is 10 nM. Concentrations above 25 nM may lead to increased background staining. Vortex and incubate in a room temperature water bath in the dark for exactly 1 hour.
4. Wash with 15 ml cold SB. The cells should be kept on ice from now on. 5. Add SB to the pellet to a final volume of 200 µl. Vortex to resuspend. Add 50 µl
Miltenyi anti-PE microbeads. Vortex again and incubate on ice in the dark for 30 min. 6. Wash with 15 ml SB. 7. While spinning, set up a Miltenyi LS column on a MidiMACS magnet and pre-rinse with
3 ml SB (discard flow-through). Place a new 15 ml conical tube under column. 8. Resuspend pellet in 3 ml SB and apply to the column, filtering through nylon mesh in the
process. 9. Rinse the original sample tube with 3 ml SB and apply to the column, also rinsing the
nylon mesh in the process. Discard nylon mesh. Add two more washes of 3 ml each to the column for a total of three x 3 ml washes. Continue collecting the flow-through into the same unbound collection tube.
10. Remove the column from the magnet and place on top of a new 15 ml conical tube. Add 5 ml SB and force the buffer through in one motion with the plunger. Discard column and plunger.
11. Spin down the bound and unbound fractions. Carefully aspirate all but about 40 µl of buffer from the bound fraction tube. Resuspend the pellet to exactly 95 µl with Fc block. Aspirate the unbound fraction tube and resuspend to 2.0 ml with Fc block. For each fraction, take out 5 µl and mix with 200 µl counting beads (Caltag-Invitrogen #PCB-100) in a 5 ml FACS tube.
12. Stain and analyze by flow cytometry immediately. For the bound fraction, add antibody cocktail directly to the remaining ~90 µl cells in the 15 ml conical tube. For the unbound fraction, remove 90 µl and stain with antibody cocktail in a 5 ml FACS tube. Incubate on ice for 30 min. Wash samples with 5 ml SB. For the bound fraction, aspirate the supernatant, resuspend the pellet in 250 µl SB, and transfer to a 1.2 ml FACS microtube (fits inside a regular 5 ml FACS tube). Rinse the 15 ml tube with another 250 µl and pool together in the same microtube. For the unbound fraction, decant the supernatant and resuspend the pellet in 2.0 ml SB.
Jenkins lab 8/4/08 2
Sample Analysis: For FACS analysis of your stained sample, use a double dumping strategy in which CD3+ events are first gated away from a lineage panel stain (B220, CD11b, CD11c, F4/80), and then CD4+ events are gated away from CD8+ events (or vice versa). CD44 has been the most reliable marker for antigen-experienced cells, as CD62L is shed during the RT tetramer incubation step, although you can use TAPI-2 inhibitor to prevent this. The following color setup is recommended: Pacific Blue: B220, CD11b, CD11c, F4/80 (optional: CD19, Gr-1, NK1.1) Pacific Orange: CD8 FITC: CD3 or Vbeta PE: tetramer PerCP: CD4 PE-Cy7: CD3 (if FITC not available) APC: CD69, CD62L or Valpha2 Alexa 700: CD44 APC-Alexa 750: I haven't found a good use for this channel yet. Always make a master mix for the antibody cocktail. If you need to comp the machine, cells from the leftover unbound fraction are great to use for comp stains because they've already been filtered away from clumpy stuff. Avoid using tandem dyes if possible. For the bound fraction, collect as many events as you can before the sample runs dry. This usually ranges between 300,000 to 2,000,000 events (varies with different lots of Miltenyi LS columns). For unbound fractions, collect 1,000,000 events. Keep the acquisition rate at around 3000 events per second. 2,000,000 total events at 3000 events per second takes about 11 minutes to run. Try to avoid collecting bubbles by stopping the acquisition before the tube runs dry. When your tube starts getting close to empty, the event rate will start to decrease. If you're collecting at around 3000 events per second, a good point to stop is when the acquisition rate falls to below 1500 events per second. Cell Count Analysis: Collect 10,000 total flow cytometry events for each of your cell count samples. Make sure you use the same machine and settings as you did for your stained cell samples. Vortex your tubes well just before acquiring your sample. It's a good idea to count your stock of counting beads on the hemacytometer once in a while to keep an accurate account of their concentration, which often changes as they get used up over time. We generally maintain our stock concentration of beads at 200,000 per ml in SB. Set a gate around the counting beads, which is best resolved by plotting FITC versus forward scatter. Then use the following formula to calculate the total number of cells in your sample:
( )total numbercells in sample
cell countbead count ( )bead vol
cell vol( )bead stockconc ( )total sample
vol= ( )total numbercells in sample
cell countbead count ( )bead vol
cell vol( )bead stockconc ( )total sample
vol=
Jenkins lab 8/4/08 3
To calculate the total number of tetramer-positive cells in your mouse, multiply the percentage of tetramer-positive events in your stained sample with the total number of cells in your bound (enriched) fraction. Here is an example (naive B6 mouse pulldown with 2W1S:I-Ab tetramer):
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SSC
-A
0 50K 100K 150K 200K 250KSSC-W
0
50K
100K
150K
200K
250K
SSC
-A
87.8
0 10 3 10 4 105
<FITC-A>: CD3
0
103
104
105
<Pac
ific
Blu
e-A
>: d
ump
5.66
0 10 3 10 4 105
<PerCP-A>: CD4
0
103
104
105
<Pac
ific
Ora
nge-
A>:
CD
8
59.5
37.3
Unbound
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SSC
-A
0 50K 100K 150K 200K 250KSSC-W
0
50K
100K
150K
200K
250K
SSC
-A
97.1
0 10 3 10 4 105
<FITC-A>: CD3
0
103
104
105
<Pac
ific
Blu
e-A
>: d
ump
29.6
0 10 3 10 4 105
<PerCP-A>: CD4
0
103
104
105<P
acifi
c O
rang
e-A
>: C
D8
57.8
38.8
0 103 104 105
<PE-A>: 2W1S:I-Ab
0
103
104
105
<Ale
xa 7
00-A
>: C
D44
7.07 0
092.9
0 103 104 105
<PE-A>: 2W1S:I-Ab
0
103
104
105
<Ale
xa 7
00-A
>: C
D44
7.63 0
092.4
0 103 104 105
<PE-A>: 2W1S:I-Ab
0
103
104
105
<Ale
xa 7
00-A
>: C
D44
8.25 9.21e-3
0.5591.2
0 103 104 105
<PE-A>: 2W1S:I-Ab
0
103
104
105
<Ale
xa 7
00-A
>: C
D44
4.67 0
095.3
CD4+ CD8+
CD4+ CD8+
85 2W1S+ events
Bound Fraction
Unbound Fraction
366,718 total events= 0.0232% 2W1S+
0 2W1S+ events
1,000,000 total events= 0% 2W1S+
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SSC
-A
0 50K 100K 150K 200K 250KSSC-W
0
50K
100K
150K
200K
250K
SSC
-A
87.8
0 10 3 10 4 105
<FITC-A>: CD3
0
103
104
105
<Pac
ific
Blu
e-A
>: d
ump
5.66
0 10 3 10 4 105
<PerCP-A>: CD4
0
103
104
105
<Pac
ific
Ora
nge-
A>:
CD
8
59.5
37.3
Unbound
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SSC
-A
0 50K 100K 150K 200K 250KSSC-W
0
50K
100K
150K
200K
250K
SSC
-A
97.1
0 10 3 10 4 105
<FITC-A>: CD3
0
103
104
105
<Pac
ific
Blu
e-A
>: d
ump
29.6
0 10 3 10 4 105
<PerCP-A>: CD4
0
103
104
105<P
acifi
c O
rang
e-A
>: C
D8
57.8
38.8
0 103 104 105
<PE-A>: 2W1S:I-Ab
0
103
104
105
<Ale
xa 7
00-A
>: C
D44
7.07 0
092.9
0 103 104 105
<PE-A>: 2W1S:I-Ab
0
103
104
105
<Ale
xa 7
00-A
>: C
D44
7.63 0
092.4
0 103 104 105
<PE-A>: 2W1S:I-Ab
0
103
104
105
<Ale
xa 7
00-A
>: C
D44
8.25 9.21e-3
0.5591.2
0 103 104 105
<PE-A>: 2W1S:I-Ab
0
103
104
105
<Ale
xa 7
00-A
>: C
D44
4.67 0
095.3
CD4+ CD8+
CD4+ CD8+
85 2W1S+ events
Bound Fraction
Unbound Fraction
366,718 total events= 0.0232% 2W1S+
0 2W1S+ events
1,000,000 total events= 0% 2W1S+
Jenkins lab 8/4/08 4
0 50K 100K 150K 200K 250KFSC-A
0
102
103
104
105
<FIT
C-A
>
55.9
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SS
C-A
0 50K 100K 150K 200K 250KFSC-A
0
102
103
104
105
<FIT
C-A
>6.42
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SS
C-A
Bound
Unbound
Bound Fraction
Unbound Fraction
5587 bead events4413 cell events
10,000 total events
642 bead events9358 cell events
10,000 total events
0 50K 100K 150K 200K 250KFSC-A
0
102
103
104
105
<FIT
C-A
>
55.9
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SS
C-A
0 50K 100K 150K 200K 250KFSC-A
0
102
103
104
105
<FIT
C-A
>6.42
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SS
C-A
Bound
Unbound
Bound Fraction
Unbound Fraction
5587 bead events4413 cell events
10,000 total events
642 bead events9358 cell events
10,000 total events
( )total numbercells in sample
cell countbead count ( )bead vol
cell vol( )bead stockconc ( )total sample
vol=
( )total numbercells in sample
44135587 ( )0.200 ml
0.005 ml( )200,000 / ml ( )0.095 ml=total number
cells in sample6.00 x 105=
Bound Fraction
( )total numbercells in sample
9358642 ( )0.200 ml
0.005 ml( )200,000 / ml ( )1.0 ml=total number
cells in sample1.17 x 108=
Unbound Fraction
total number 2W1S+cells in mouse
percentage 2W1S+cells in bound fraction
total number cellsin bound fraction= x
total number 2W1S+cells in mouse
0.0232% 6.00 x 105= xtotal number 2W1S+
cells in mouse139=
( )total numbercells in sample
cell countbead count ( )bead vol
cell vol( )bead stockconc ( )total sample
vol=
( )total numbercells in sample
44135587 ( )0.200 ml
0.005 ml( )200,000 / ml ( )0.095 ml=total number
cells in sample6.00 x 105=
Bound Fraction
( )total numbercells in sample
9358642 ( )0.200 ml
0.005 ml( )200,000 / ml ( )1.0 ml= ( )total numbercells in sample
9358642 ( )0.200 ml
0.005 ml( )200,000 / ml ( )1.0 ml=total number
cells in sample1.17 x 108=
Unbound Fraction
total number 2W1S+cells in mouse
percentage 2W1S+cells in bound fraction
total number cellsin bound fraction= x
total number 2W1S+cells in mouse
0.0232% 6.00 x 105= xtotal number 2W1S+
cells in mouse139=
Jenkins lab 8/4/08 5
Notes: 1) If you have a very large population of antigen-specific cells in your sample, you should
also calculate the number of tetramer-positive cells in your unbound fraction, and add this to your total for the mouse. When the number of tetramer-specific cells in your sample is very large, for example if you're analyzing a mouse at the peak of a T cell response to an infection, the efficiency of the tetramer pulldown can be rather low, sometimes as low as 60%.
2) The fix/perm procedure for FoxP3 staining using the eBioscience kit works well with
tetramer stained cells. Remember to fix your comp samples too. A problem with fixing or perming cells is that your forward vs. side scatter properties will change dramatically, and your red cells will lyse. Therefore, your bead count samples will look very different from your samples for analysis. To deal with this, draw a forward vs. side gate in your bead count sample on what you think is equivalent to the total cell population in your analysis sample (exclude red cells and very small stuff). Then use this number instead of all non-bead events as your cell event number in your calculations.
