Jenkins lab 1 8/18/15  

Jenkins Lab MHCII Tetramer Pulldown Protocol using EasySep from Stemcell Technologies - 8/18/15

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 sorter buffer (PBS + 2% FBS, 0.1% NaN3) 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 (and spin down (Beckman tabletop centrifuge 1,600 rpm, 5 min 4oC).

a. Alternatively, harvest spleen and lymph nodes (the spleen needs to be chopped up into smaller pieces, approximately lymph node size) into a gentleMACS c-tube containing 3 mL of ice-cold sorter buffer. Place tube in gentleMACS Octo Dissociator from Miltenyi Biotec and run on Spl-4. Check the tube after dissociation and run tube on Spl-4 again if there are still chunks of spleen that have not been dissociated. Top up with 10 mL cold sorter buffer (and spin down (Beckman tabletop centrifuge 1,600 rpm, 5 min 4oC). Then resuspend pellet in 5 mL of ice-cold sorter buffer and filter into a 15 ml polypropylene conical centrifuge tube. Rinse original tube with 5 mL ice-cold sorter buffer (and spin down (Beckman tabletop centrifuge 1,600 rpm, 5 min 4oC).

2. Aspirate and add Fc block (2.4G2 SFM supernatant + 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.

2. Add tetramer to the sample at a final concentration of 10 nM, depending on the dose response curve for the current production lot of tetramer. Do not vortex the stock tube of tetramer. Vortex the sample tube and incubate at room temperature in the dark for 1 hour.

a. If staining for chemokine receptors, like CXCR5, then add the antibody at the same time as the tetramer for optimal staining (room temperature in the dark for an hour).

4. Wash with 15 ml cold sorter buffer (SB). 5. Add SB to the pellet to a final volume of 500 µl. Vortex to resuspend. Add 6.25 µl

Stemcell anti-fluorophore antibody cocktail (use antibody cocktail specific for the fluorophore on the tetramer). Vortex again and incubate at room temperature in the dark for 15 min.

6. Mix Stemcell Easysep Magnetic particles for positive selection with pipette (do not vortex) and add 25 uL of particles per sample tube. Vortex and incubate at room temperature in the dark for 10 min.

7. Resuspend the samples to a final volume of 2.5 mL and transfer to a FACS tube, filtering through nylon mesh in the process.

8. Vortex samples on a very low setting (2-3 on a Vortex-genie 2) and place in EasySep magnet for 5 minutes. During the 5 minute incubation, set up collection tubes for the unbound fraction (round bottom 13 mL 100 X 16 mm polypropylene work well for this purpose when using the EasySep magnets in an EasySep multistand). Decant sample tubes by pouring the sample tubes while they are still in the magnet into the collection

Jenkins lab 1 8/18/15  

tubes . 9. Wash the sample to improve the purity by removing sample tubes from the magnets and

adding 2.5 mL ice cold sorter buffer. Vortex samples on a very low setting (2-3 on a Vortex-genie 2) and place in EasySep magnet for 5 minutes. Decant as described previously. Repeat two more times for a total of three washes.

10. After the final wash, remove the FACS tube containing the bound cells from the magnet. Spin down the bound and unbound fractions. Carefully pour out the buffer from the bound fraction tube. Resuspend the pellet to exactly 95 µl with Fc block. Pour out the buffer from the unbound fraction tube and resuspend to 2.0 ml with Fc block. For each fraction, take out 5 µl and mix with 100 µl counting beads (AccuCheck counting beads from Life technologies #PCB100 diluted to 200000 beads/mL) in a 5 ml FACS tube.

11. 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 2 8/18/15  

Sample Analysis:  For FACS analysis of your stained sample, use a double dumping strategy in which CD90.2+ 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 can be shed during the tetramer incubation step. The following color setup is recommended as a starting point:  

APC-eflour780: B220, CD11b, CD11c, F4/80, Tonbo Ghost dye 780 live/dead PE: tetramer BV786: CD4 FITC: CD8a APC: tetramer Alexa 700: CD90.2 (this is very bright so dilute to 1:300) BV510 or V500: CD44

The following color setup is recommended for analyzing T follicular helper cells:  

APC-eflour780: B220, CD11b, CD11c, F4/80, Tonbo Ghost dye 780 live/dead PE: tetramer BV786: CD4 FITC: CD8a APC: tetramer Alexa 700: CD90.2 (this is very bright so dilute to 1:300) BV510 or V500: CD44 BUV395: CXCR5 PE-Cy7: PD-1

The following color setup is recommended for analyzing transcription factors in T cells:  

PE: tetramer AF488: Bcl6 (1:20) APC: tetramer Alexa 700: CD90.2 (this is very bright so dilute to 1:300) APC-eflour780: B220, CD11b, CD11c, F4/80, Tonbo Ghost dye 780 live/dead BUV395: CXCR5 Percp-Cy5.5: Foxp3 BV421: Rorgt V500: CD44 BV605: T-bet (1:80) BV786: CD4

 Always make a master mix for the antibody cocktail. Cells left over from the unbound fraction are great to use for compensatoin stains because they have already been filtered away from clumpy stuff (a great alternative to cells are the ultracomp ebeads from eBioscience).

Jenkins lab 2 8/18/15  

=

 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. For unbound fractions, collect 1,000,000 events. Keep the acquisition rate at around 3,000 events per second. 2,000,000 total events at 3,000 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 3,000 events per second, a good point to stop is when the acquisition rate falls to below 1,500 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:

   

cells in sample

total number

cells in sample cell count

bead count bead stock conc

bead vol cell vol

total sample vol

Jenkins lab 3 8/18/15  

Unbound

250K 250K 10 10

 200K 200K 8

dump104 D10

C

 SSC-A SSC-A Blue-A>:

103

Orange-A>:

37.3 150K 150K

10

100K 100K 59.5 5.66

250K 250K 10 10

 200K 200K 8

dump104 D10

C

 SSC-A SSC-A Blue-A>:

103

Orange-A>:

38.8 150K 150K

10

100K 100K

<Pacific 57.8

97.1 29.6 0

7.07 0 7.63 0 105 10

 4104 410 4 4

D D C C  

700-A>:10

700-

A>:10

 <Alexa <Alexa

0 0  

 92.9 0 92.4 0

0 103 104 105 0 103 104 10

8.25 9.21e-3 4.67 0 105 10

 4104 410 4 4

D D C C  

700-A>:10

700-

A>:10

 <Alexa <Alexa

0 0  

 91.2 0.55 95.3 0

0 103 104 105 0 103 104 10

Unbound

SS

C-A

S

SC

-A

SS

C-A

S

SC

-A

<Pac

ific

Blu

e-A

>: d

ump

<Pac

ific

Blu

e-A

>: d

ump

<Ale

xa 7

00-A

>: C

D44

<A

lexa

700

-A>:

CD

44

<Pac

ific

Ora

nge-

A>:

CD

8 <P

acifi

c O

rang

e-A

>: C

D8

<Ale

xa 7

00-A

>: C

D44

<A

lexa

700

-A>:

CD

44

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 2W:I-Ab tetramer). We now use CD90.2 in place of CD3e because it is brighter, but all of the gating and calculations are the same:

 Bound Fraction

 250K 250K

105 105

 200K

 150K

200K

 150K

 104

 103

 104

   103

 37.3

100K

 50K

100K

 50K

 87.8

 5.66 0

0

 59.5

 0

0 50K 100K 150K 200K 250K FSC-A

0

0 50K 100K 150K 200K 250K SSC-W

 0 10 3 10 4 105

<FITC-A>: CD3

 0 10 3 10 4 105

<PerCP-A>: CD4

 CD4+ CD8+

 105

 8.25 9.21e-3

 105

 4.67 0

     

85 2W1S+ events  

366,718 total events

     = 0.0232% 2W+

104

 103

   

0

104

 103

   

0

 91.2 0.55

0 103 104 105

<PE-A>: 2W1S:I-Ab

95.3 0

0 103 104 105

<PE-A>: 2W1S:I-Ab

 Unbound Fraction

 250K 250K

105 105

 200K

 150K

 100K

 50K

200K

 150K

 100K

 50K

         97.1

 104

 103

   

0

         29.6

 104

   103

 

0

   38.8

       57.8

 0

0 50K 100K 150K 200K 250K FSC-A

0

0 50K 100K 150K 200K 250K SSC-W

 0 10 3 10 4 105

<FITC-A>: CD3

 0 10 3 10 4 105

<PerCP-A>: CD4

 CD4+ CD8+

 105

 7.07 0

 105

 7.63 0

 104 104

 0 2W1S+ events

 

1,000,000 total events

 = 0% 2W+

 103

   

0

 103

   

0

 92.9 0

0 103 104 105 <PE-A>: 2W1S:I-Ab

92.4 0

0 103 104 105 <PE-A>: 2W1S:I-Ab

Jenkins lab 4 8/18/15  

 

   

55.9

   

6.42

( )( )( )( )( )

)( )( 0.005 ml )(

SS

C-A

S

SC

-A

<FIT

C-A

> <F

ITC

-A>

= )( )( ) = (

= (

= x =

=

Bound Fraction  

250K  105

 200K

   

150K    

100K    

50K

   104

   103

   102

 

0

 55.9

   

5587 bead events 4413 cell events

 

10,000 total events

 0

0 50K 100K 150K 200K 250K FSC-A

   0 50K 100K 150K 200K 250K

FSC-A

 Unbound Fraction

 250K  

105

 200K

   

150K    

100K    

50K

   104

   103

   102

 

0

 6.42

   

642 bead events 9358 cell events

 

10,000 total events

 0

0 50K 100K 150K 200K 250K FSC-A

   0 50K 100K 150K 200K 250K

FSC-A

   

Bound Fraction  

total number cells in sample

   

cell count bead count

   bead stock

conc

   bead vol cell vol

   total sample

vol  

total number cells in sample

 4413 5587

 

200,000 / ml 0.200 ml 0.095 ml 0.005 ml  

total number cells in sample

 

= 6.00 x 105

 Unbound Fraction

 

total number cells in sample

   

9358 642

   

0.200 ml 200,000 / ml 0.005 ml

 

1.0 ml )  

total number cells in sample

 

= 1.17 x 108

 total number 2W1S+

cells in mouse percentage 2W1S+

cells in bound fraction total number cells in bound fraction

 

total number 2W1S+ cells in mouse

 

total number 2W1S+ cells in mouse

0.0232% x  

 139

 6.00 x 105

Jenkins lab 5 8/18/15  

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 (This is especially true for examining PE-specific B cells, which have a very large naïve population). 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. Also, it is best to spin your cells at 2100 rpm instead of 1600 rpm after fixation to reduce cell loss during wash steps (it is okay to spin fixed cells faster because it won’t effect their viability, their dead). 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.

3) When determining the naïve cell number for a given tetramer, we recommend double

tetramer staining in APC and PE. For example, we stain with the 2W:I-Ab APC and 2W:I-Ab PE tetramers at their normal amounts (10 nM final concentration) when we are determining the naïve precursor frequency of the 2W:I-Ab specific population. It is also important to enrich for both fluorophores by adding the standard amount (6.25 uL) of the anti-APC cocktail and the anti-PE cocktail from Stemcell Technologies.