Hanks’ balanced salt solution, preparation

Percoll Gradient Preparation Percoll Stock Mix 180 mL Percoll (Pharmacia, Piscataway, NJ), 18 mL 10X Hanks Balanced Salt Solution (HBSS) without Ca,... 

Hanks balanced salt solution (HBSS), pH 7.4. Prepare a IXsolutionfiom the lOX concentrate (obt ned from Gibco, Paisley, Scotland) and adjust the pH with 1.76 mL of 7.5% NaHCOj/lOOmLWarm to37°C (seeNote 3). 

Sample preparation 100 pL Cell suspension -I- 100 pL cefoperazone solution -I- 100 pL Hanks balanced salt solution, sonicate 30 min, add 800 pL MeCN, centrifuge at 13000 g... 

Tissue Preparation 1. P2 Sprague-Dawley Rat pups. 2. Dulbecco Modified Eagle s Medium (DMEM), high glucose, no glutamine, no pyruvate (Cambrex). 3. Ca +-Mg free Hanks Balanced Salt Solution (Cambrex). 4. 2.5% trypsin solution (Cambrex) diluted 1 10 in HBSS for 0.25% final, aliquot and store at -20°C, stable for 1 year. 5. DNAse 1 (Boehringer Mannheim) prepare 1 mg/mL stock in PBS aliquot store at -20°C stable for 1 year. 6. Heat-inactivated FBS (Cambrex) aliquot and store at -20°C. 

Prepare daily HBSS/BSA 10 mL sterile 10X Hank s balanced salt solution (HBSS, with phenol red, without Ca2+/Mg2+ Life Technologies, Bethesda, MD),... 

Figures 8.1 and 8.2 show intradermal and mammary fat pad injections, respectively. For photographic purposes, the animals were also sedated for intradermal injections. Both types of injection used 27 gauge needles attached to tuberculin 1-cc syringes. The singlecell suspension was prepared in ice-cold Hank s Balanced Salt Solution (however, any isotonic hquid will work as long as it does not contain serum). Note that the bevel faces the syringe markings, making it easy to see injection volume. Also note that for all of the injections the bevel is oriented so that the bore is visible from the top.

PMN and mononuclear leukocytes (MNL) were prepared from Ficoll-Hypaque density difference centrifugation as previously described by English (1976). After isolation, the cells were enumerated and diluted to desired concentrations in Hank s balanced salt solution (BBSS), Grand Island Biological Company, Grand Island, New York. 

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