Lymphocytes Antibody

Rabbit Anti-Mouse Lymphocyte Serum

Cat. Num. AAA524030

• Synonyms: Lymphocytes; Rabbit Anti-Mouse Lymphocyte Serum; Lyophilized (Polyclonal) (rabbit serum); anti-Lymphocytes antibody

• Presentation: 1.0 ml, 5.0 ml lyophilized
• Sterility: This reagent is not sold as sterile, but can be sterilized by filtration if necessary. To minimize loss of volume during filtration, dilute to the final working concentration in the appropriate medium before filtration and filter through a 0.22um filter.
• Specifications: Immunizing Strain: BALB/c; Heat Inactivation: 60 minutes at 56°C; Absorption: Mouse erythrocyes/hepatocytes
• Lot Specifications: Antiserum Titration:; Cell Source: Spleen, Thymus; Donors: BALB/c; Cell Concentration: 1.1x10⁶ cells per ml; Complement: Low-Tox-M Rabbit Complement; Complement Concentration: 1:10; Procedure: Two stage cytotoxicity as described on Recommended Method for Determining Percent Cytotoxicity with Anti-Mouse Lymphocyte Serum Plus Complement.
• Tissue Distribution: Procedure: As above; Antiserum Concentration: 1:80; Strain: BALB/c
• Cell Source/C.I.: Thymus: 100.0; Spleen: 50.0; Lymph Node: 95.0; Bone Marrow: 81.0; ; Antiserum dilution that results in 50% cytotoxicity against thymus cells: 1:1350
• Strain Distribution: Target Cell: Thymus; Procedure: As above
• Strains Tested (+/-): C57BL/6: -; C3H/He: -; ATH: -; A.TL: -; CBA: -; BALB/c: +
• Functional Testing: Cell Source: Splenocytes and Thymocytes; Cell Concentration: 1x10⁶ cells/ml.; Antiserum Concentration: 1:10; Complement: Low-Tox-M at 1:10; Donors: BALB/c; ; Procedure: Cells were treated as described in "Recommended Method for Depleting A Cell Population of Mouse Lymphocytes." The remaining viable cells were exposed to the mitogens Concanavalin A (CON A), Phytohaemagglutinin (PHA), and Lipopolysaccharide (LPS). Cell depletionwith Anti-Mouse Lymphocyte Serum was found to inhibit the CON A, PHA,and LPS responses. Treatment of mouse splenocytes with Anti-Mouse Lymphocyte Serum plus complement essentially eliminated in vitro T effector cell function.
• Recommended Method For Depleting A Cell Population Of Mouse Lymphocytes: 1. Prepare a cell suspension from the appropriate tissue in Cytotoxicity Medium or equivalent. Remove red cells and dead cells (where necessary) by purification of viable lymphocytes on Lympholyte-M² density cell separation medium. After washing, adjust the cell concentration to 1x10⁶ cells per ml in Cytotoxicity Medium.; 2. Add the antiserum to a final concentration of 1:40 and mix.; 3. Incubate for 60 minutes at 4°C.; 4. Centrifuge to pellet the cells and discard the supernatant.; 5. Resuspend to the original volume in Low-Tox-M³ Rabbit Complement, diluted to the appropriate concentration in Cyotoxicity Medium. (Recommended concentration included with each batch of Low-Tox-M Complement); 6. Incubate for 60 minutes at 37°C.; 7. Monitor for percent cytotoxicity at this stage, before further processing. For this purpose remove a small sample from each tube, dilute 1:10 with medium, and add 1/10 volume of 1% Trypan Blue. After 3-5 minutes, score live versus dead cells in a hemacytometer.; 8. For functional studies, remove the dead cells from the treated groups before further processing, particularly if the treated cells are to be cultured. Layering the suspension on cell separation medium and centrifuging at room temperature as per the instructions provided can do this. Live cells will form a layer at the interface, while dead cells pellet.The interface can then be collected and washed in Cytotoxicity Medium before being resuspended in the appropriate medium for further processing.; ; Alternatively, the cells can then be washed and resuspended in the appropriate medium for further processing immediately after Step #6, provided that the dead cells will not interfere with subsequent assays.
• Recommended Method For Determining Percent Cytotoxicity With Anti-Mouse Lymphocyte Serum Plus Complement: 1. Prepare a cell suspension from the appropriate tissue in Cytotoxicity Medium or equivalent. Remove red cells and dead cells (where necessary) by purification of viable lymphocytes on Lympholyteo-M² density cell separation medium. After washing, adjust the cell concentration to 1.1 x 10⁶ cells per ml in Cytotoxicity Medium.; 2. Add the antiserum to a final concentration of 1:40 and mix.; 3. Incubate for 60 minutes at 4°C.; 4. Centrifuge to pellet the cells and discard the supernatant.; 5. Resuspend to the original volume in Low-Tox-M Rabbit Complement³ diluted to the appropriate concentration in Cyotoxicity Medium. (Recommended concentration included with each batch of Low-Tox-M Rabbit Complement ~ 1:10 - 1:25); 6. Incubate for 60 minutes at 37°C.; 7. Place on ice.; 8. Add Trypan Blue. 10% by volume of 1%Trypan blue (w/v) added 3-5 minutes before scoring works well. Score live versus dead cells in a hemacytometer. ; Cyotoxic index (C.I.) can be calculated as follows: ; C.I. = ^{% cyt (antibody + complement) - % cvt (complement alone)}/_{100% - % cyt (complement alone)} x 100
• Preparation and Storage: Lyophilized form stable at 4°C or -20°C. Reconstitute with 1.0 ml or 5.0 ml distilled water. ; After reconstitution, aliquot and freeze unused portions at -70°C in volumes appropriate for single usage. ; Avoid repeated freeze/thaw cycles.

Testing Data

Related Product Information for anti-Lymphocytes antibody

Description: Anti-Mouse Lymphocyte Serum is prepared by immunizing rabbits with mouse thymus, spleen and lymph node cells followed by absorption with mouse erythrocytes and hepatocytes. This antiserum is strongly cytotoxic to all mouse lymphocytes.

Notes: 1. Cytotoxicity Medium is RPMI-1640 with 25mM Hepes buffer and 0.3% bovine serum albumin (BSA). BSA is substituted for the conventionally used fetal calf serum (FCS) because we have found that many batches of FCS contain complement dependent cytotoxins to mouse lymphocytes, thus increasing the background killing in the presence ofcomplement. We recommend that cells not be exposed to FCS prior to or during exposure to antibody and complement. Some batches of BSA als ocontain complement dependent cytotoxins, resulting in the same problem. We screen for batches of BSA giving low background in the presence ofcomplement and use the selected BSA for preparing Cytotoxicity Medium.

2. Lympholyte-M cell separation medium is density cell separation medium designed specifically for the isolation of viable mouse lymphocytes. This separation medium provides a high and non-selective recovery of viable mouse lymphocytes, removing red cells and dead cells. The density of this medium is 1.0865-1.0885. Isolation of mouse lymphocytes on cell separation medium of density 1.077 will result in high and selective loss of lymphocytes and should be avoided.

3. Rabbit serum provides the most potent source of complement for use with antibodies to mouse cell surface antigens. However, rabbit serum itself is very toxic to murine lymphocytes. Low-Tox-M Rabbit Complement is absorbed to remove toxicity to mouse lymphocytes, while maintaining its high complement activity. When used in conjunction with Cytotoxicity Medium, this reagent provides a highly potent source of complement with minimal background toxicity.