Gut Tropic Competitive Homing Assay, Villablanca EJ, & Mora JR., J. Vis Exp, 2011
1) Isolate lymphocytes from donor mice, need 3x106/recipient for each cell type minimum – depending on cell type and organ (for homing efficiencies to various organs, see e.g. Halin et al., the S1P1-competent control cells, Blood 2005)
2) Stocks of TRITC and 5(6)-FAM-SE (“green CFSE”) in water or PBS, kept at –20°C; label cells as follows:
5(6)-FAM-SE (yellow color, “green CFSE”, MW=473.39), for homing experiments (Stock: 10 mg/ml (20mM) in DMSO)- For labelling: 0.5-3.5 ml 5(6)FAM-SE stock/1ml labeling buffer (RPMI 2% FCS) + max 15x106 cells, 15min/37°C water bath----underlay with FBS and spin, resuspend pellet, inject.
TRITC, for homing experiments (Molecular Probes T-1481, R-isomer, MW 433.52 (544/572 nm)) Stock: 30 mg/ml (most commonly; variations may depend on solubility of the batch:
Dissolve 5 mg in 50 ml PBS. Rock at RT, protecting from light for 60’ or more, Filter twice through 0.45 µm and then 0.2 µm filters, Make serial dilutions in PBS : 1:5 and 1:2 dilutions henceforth, Measure absorbance at 550 nm to determine end concentration. (Absorption coefficient 85000 M-1cm-1), MW = 444 g/mol-- Concentration (M) x 444 = Concentration (mg/mL)
For labelling: 20ml stock/1ml for 20min/37°C water bath in labelling buffer (RPMI 2% FCS)- (alternatively 30-35ml stock/1ml for 10-15min/37°C), underlay with FBS and spin, resuspend pellet, inject.
4) Read-out at FACS; TRITC may bleed into FL-3, but counterstain with APC (FL-4) is possible.
*For IEL medium and HBSS+Hepes, see recipes at the end.
1) Dissect small (SI) and large intestine (LI).
2) Place the SI and LI in a 10 mm Petri dish with cold HBSS. Flush the SI with 20 ml cold HBSS using a 20 ml syringe and a round-tip feeding needle.
3) Remove the fat as much as possible using forceps.
4) Carefully remove ALL the Peyer’s patches (cut them beyond their border to make sure that no remaining lymphoid tissue is left).
5) Cut the SI longitudinally and then into 0.5-1.0 cm pieces.
6) Place the SI pieces in a 50 ml tube with 20 ml cold HBSS and leave on ice. If you are not isolating LI lymphocytes, proceed to step 11.
7) For isolation of LI lymphocytes, dissect out the appendix/cecum (usually 1.0-1.5 cm from the cecum’s tip should be enough). Remove the fat as much as possible using forceps.
8) Cut longitudinally and then wash the stools out with a syringe filled with cold HBSS using a feeding needle.
9)Cut the LI into 0.5-1.0 cm pieces and put them in a 50 ml tube with 20 ml cold HBSS.
10)Wash three times SI and/or LI with 20 ml cold HBSS (filter SI or LI every time through a metal ‘tea’ strainer).
11)Add 25 ml IEL medium at 37oC and incubate with shaking (150/min) for 30 min at 37oC.
12)After that, shake vigorously for 25-30 sec (as hard as possible !!!).
13)Filter SI and/or LI plus the supernatant (SN) through a 70 mm cell strainer (B&D, Falcon) into a 50 ml tube. SAVE the SN and put the tube on ice (it contains most of the IEL fraction).
14)Optional: Collect the SI and/or LI pieces, add 25 ml IEL medium and repeat steps 13-14. If you are not isolating lamina propria (LP) lymphocytes, proceed to step 18.
15)For isolating LP lymphocytes, take the remaining tissue (from SI and/or LI) and incubate it in 5 ml 400 U/ml Collagenase-D+50 mg/ml DNAse-I for 30-40 min at 37oC (in a rocker).
16)After that, filter the LP and SN through a 70 mm cell strainer, mincing the tissue with a syringe plunge and adding IEL medium up to 30 ml.
17)Centrifuge (300 x g, 5 min, 4oC) IEL and/or LP cell suspensions.
18) Discard SN and resuspend the pellets in 4.5 ml 44% Percoll. Vortex briefly and put the cells in a 15 ml tube.
19) Very carefully, underlaid 2.3 ml 67% Percoll (using a 2 ml pipet). Two distinct phases should be clearly visible and delimited.
20)Important: In order to obtain a better separation of the cells, use the Percoll solutions at RT (20oC).
21)Centrifuge at 600 x g for 20 min, 20oC with smooth acceleration and NO brake.
22)If the gradient was successful, the lymphoid fractions should be visible as a turbid ring in the 44%-67% Percoll interphase. The epithelial cells (and other low-density cells) will float on top of the 44% layer and erythrocytes, dead cells, and debris should be in the pellet.
23)Carefully remove the epithelial and other low-density stuff with a Pasteur pipet and gentle aspiration. Collect the lymphoid fraction from the 44%-67% interphase using a 2 ml pipet and put it in a new 15 ml tube (about 3-4 ml).
24)Add IEL medium up to 12 ml. Centrifuge (300 x g, 5 min, 4oC).
25)Resuspend the IEL and/or LP cells in your culture medium.
HBSS: 500 ml Hanks balanced salt solution without Ca++/Mg++ (1x) + 10 ml 1M Hepes buffer + 5 ml 100x penicillin/streptomycin + 0.25 ml gentamicin (40 mg/ml)
IEL Medium: 1000 ml RPMI + 20 ml FBS + 20 ml 1M Hepes buffer + 10 ml 100x penicillin/streptomycin + 0.50 ml gentamicin (40 mg/ml)
100% Percoll (Final pH should be approx. 7.2. Stable up to 1 month at 4oC)
For 50 ml add: 45 ml stock Percoll + 4.48 ml HBSS w/o Ca++/Mg++ (10x) + 0.50 ml 1 M Hepes buffer + 0.23 ml 1 N HCl (Sigma, already prepared)
Percoll quantities required for 1 mouse (if isolating LP and IEL from SB and colon), 2 mice (if only isolating IEL from LP and colon), or 4 mice (if only isolating IEL from SB):
67% Percoll (72% if isolating neutrophils) 6.6 ml 100% Percoll 3.4 ml HBSS
44% Percoll 8.80 ml 100% Percoll, 11.2 ml HBSS
*67% and 44% Percoll should be freshly prepared every time and used at RT (20 oC).