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7 labeled cells per mouse in 0.5 ml of HBSS with or without the addition of blocking reagent. Depletions were done as above. Cells treated with whole antibody or Fab fragments were incubated in a saturating concentration of antibody for 15 min on ice and then washed before injection. HA and chondroitin sulfate (Sigma) were added to the cell suspension at the time of injection (final concentration, 0.5 μM). Hyaluronidase (ICN Biochemicals) and chondroitinase ABC (Sigma) treatment was done by injecting 10 U per mouse iv 30 min before donor cell infusion. Ninety minutes after the infusion of labeled cells, the recipient mice were killed and PELs were analyzed for the homing of labeled cells.
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note
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3, and 0.1 mM EDTA. PELs were collected by peritoneal lavage using 5 ml of RPMI (37°C) containing 2% FBS and 2 mM EDTA. Staining of PELs was performed as above, except cells were preincubated with anti-CD32/CD16 (clone 2.4G2) and stained in the presence of 10% normal mouse serum to inhibit Fc receptor interactions. Data were collected on a FACScan analytical instrument (Becton Dickinson) and analyzed using Lysis II software.
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We thank M. Kosfiszer for technical assistance and L. Picker for helpful discussions. Supported by grants from NIH (R01 CA57471 and HL56746), the Arthritis Foundation, and the Welch Foundation (I-1227). M.H.S. is an Established Investigator of the American Heart Association. H.C.D. is supported by an NIH Cancer Immunology Training Grant.
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