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T CELL ISOLATION

ISOLATION OF T CELLS FROM SYNOVIAL MEMBRANES

USE STERILE TECHNIQUES
Initial Cell Isolation:
  1. Cut away fat and dense collagenous tissue from the synovial membrane sample.
  2. Mince the remaining tissue into small pieces (a few mm in size) with scissors. Resuspend these pieces in a 0.5% trypsin solution for about 1 hour at 37°C in a shaking incubator. At the end of this time, add fresh trypsin solution and digest for another hour. Longer periods of digestion result in tissue that clogs the cell sieve.
  3. Pour this solut ion through an autoclaved cell sieve into a Petri dish. Push the remaining tissue against the sieve with the rubber end of a syringe plunger to harvest more cells. This technique will produce close to a single cell suspension. Centrifuge the cells once, resuspend them in RPMI-1640 + L-glutamine + pen/strep, with 10% fetal calf (or human) sera, and transfer them to a Petri dish. Incubate for 12 - 24 hours in a 37°C in a 5% CO2 incubator.
  4. Collect the nonadherent cells from the Petri dish, centrifuge once and replate as above. Add fresh media to adherent cells and incubate for another 24 hours.
  5. Collect and centrifuge the nonadherent and adherent cells in separate tubes. Resuspend in 9 ml of balanced salt solution (BSS), PBS or medium.
Nonadherent Cell Isolation: Carefully layer the nonadherent cells on 3 ml of ficoll-paque. Centrifuge for 35 minutes, 18-20°C at 400 xg (1400 rpm in the IEC PR-J or 1500 rpm in the Beckman TJ-6 centrifuge). Collect the banded cells, dilute in BSS and centrifuge to pellet the cells. (If red blood cells are present, resuspend the cells in 1 ml of NH4Cl erythrocyte lysing solution and incubate at 37°C for 6 minutes. Dilute the cell suspension with 5 - 10 ml of BSS and centrifuge the cells.) Resuspend the cells in 1 ml of BSS. Dilute an aliquot 1/20 in 2% acetic acid and count in a hemocytometer. Adjust the cell concentration with BSS to = 106 cells/ml. Add 1/10 of this volume as neuraminidase treated sheep red blood cells (SRBC). Centrifuge at 400 xg (1500 rpm in the Beckman TJ-6) for 5 minutes to gently pellet the cells. Let the cells sit on ice for 1 hour to form E rosettes. Very gently resuspend the cells, layer them on ficoll-paque and centrifuge as above. The pelleted cells contain the SRBC E+ rosette forming cells (i.e . T cells), while the banded cells are E- rosette forming cells. Wash each group of cells in BSS, lyse the SRBC with the NH4Cl erythrocyte lysing solution, and wash the cells again in BSS. The E- rosette cells may be kept separately, or combined with the adherent cells and the cells that pelleted out of the first ficoll-paque gradient at the beginning of this paragraph. For preparation of DNA from the T cells, the nonadherent E+ cells are now ready for resuspension in Tris-buffered saline as described in the pulse-field gel electrophoresis protocol.

Adherent Cell Isolation: Remove the adherent cells from the Petri dish with vigorous pipeting. Wash once with BSS. Save these cells and add to them the pelleted cells from the first ficoll-paque gradient described in paragraph 5a. (These are the adherent cells plus nonadherent pelleted cells.) E- rosette forming cells from the second ficoll-paque gradient in paragraph 5a may also be added to these, making them a collection of all E- cells. Lyse red blood cells that may be present with one or more treatments of NH4Cl erythrocyte lysing solution. For DNA isolation, refer to the genomic DNA isolation protocol, or to the pulse field gel electrophoresis protocol for small numbers of cells.


RECIPES

Neuraminidase-Treated Sheep Red Blood Cells
Centrifuge 20 ml of sheep red blood cells at 2000 rpm for 10 minutes at room temperature. Resuspend the cells in 20 ml of RPMI-1640. Dilute Vibrio cholera neuraminidase (Behring Diagnostics) 1:10 in RPMI. To 5 ml of centrifuge-packed sheep red blood cells, add 1 ml of dilute neuraminidase and 19 ml of RPMI-1640. Invert to mix. Incubate at 37°C for 60 minutes, inverting a few times at approximately 10 minute intervals to keep the cells resuspended. Wash the cells three times in RPMI-1640 and resuspend in 50 ml of RPMI-1640 with 10% fetal calf serum and pen/strep. Store at 4°C. These cells are good for 2 weeks.

Erythrocyte Lysing Solution
0.83 g NH4Cl
0.1 g KHCO3
90 µl of 10% EDTA (optionally added as a preservative)
QS to 100 ml with water. Filter sterilize, and store at room temperature
Balance Salt Solution (BSS)
Stock Solution 1
3.0 g dextrose
0.6 g KH2PO2(H2O)7
1.07 g Na2HPO4(H2O)7
6 ml 0.5% phenol red so lution
Dissolve and bring to 300 ml with double distilled water.

Stock Solution 2
558 mg CaCl2(H2O)2
1.2 g KCl
24 g NaCl
0.6 g MgCl2(H2O)6
0.6 g MgSO4(H2 O)7
Dissolve and bring to 300 ml with double distilled water. Let the stock solutions stand overnight at 4°C. They may be stored at 4°C or at room temperature. The pH of stock solutions should be around 7.2.

To prepare 3 L of BSS, add 300 ml of stock solution 1 to 2.4 L of water and mix. Add 300 ml of stock solution 2 to the dilute stock solution 1 and mix. Filter sterilize. Incubate for 1 week to check for contamina tion. Store at room temperature.
Tris-buffered Saline
50 mM Tris-HCl, pH 8.0
150 mM NaCl
    Send comments and updates to  Dr. Bart Frank, Arthritis and Immunology Program, OMRF
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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Isolation of T Cells from Synovial Membranes. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/tcelliso.html). 1997. Oklahoma City. Revisio n Date: October 2, 1997."