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Anti Rabbit RT Monoclonal Antibody Ascites

VIEWS: 6 PAGES: 3

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                                     Anti-Rabbit RT2
                                Monoclonal Antibody - Ascites
CL8802A
LOT: 002
DESCRIPTION:
A monoclonal antibody directed against an antigen (RT2) found on the majority of rabbit T cells and a proportion of
polymorphonuclear leukocytes.

PRESENTATION:
0.5 ml, frozen ascites.

STORAGE/STABILITY:
Store at –70°C. After thawing at room temperature, aliquot and freeze unused portions in volumes appropriate for
single usage. Do not dilute prior to freezing. Store frozen aliquots at –70°C. Avoid repeated freeze/thaw cycle.
This antibody is not sold as sterile, but can be sterilized by filtration if necessary.

METHODS FOR USE IN RABBIT T CELLS:

Recommended Method for Depleting A Rabbit Cell Population of RT2 Bearing Cells:

1.  Prepare a cell suspension from the appropriate tissue in Cedarlane Cytotoxicity Mediuma (CL95100) or
    equivalent. Remove red cells (where necessary) by treating cell pellet with 5 times the volume of 0.17M
    NH4Cl-Tris Buffer for 3 minutes at room temperature or by purification on cell separation medium. After
    washing, adjust cells to 1 x 107 cells/ml in Cytotoxicity Mediuma.
2. Add the RT2 antibody to a final concentration of 1:6 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 Cytotoxicity Medium with the appropriate concentration of Cedarlane
    Baby Rabbit Complement (CL3441)b (a final dilution of 1:1)
 6. Incubate for 60 minutes at 37°C.
7. Place on ice.
                                                                                         Continued overleaf…
8.   Monitor for cytotoxicity RE: remove a small sample from each tube, dilute 1:10 and 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.
9. For functional studies, remove dead cells from treated groups before
    processing, particularly if the treated cells are to be cultured.

Recommended Method for Determining % of RT2 Bearing Cells in a Rabbit Cell Population:

1.  Prepare a cell suspension from the appropriate tissue in Cedarlane Cytotoxicity Mediuma (CL95100) or
    equivalent. Remove red cells (where necessary) by treating cell pellet with 5 times the volume of 0.17M
    NH4Cl-Tris Buffer for 3 minutes at room temperature or by purification on cell separation medium. After
    washing, adjust cells to 1 x 106 cells/ml in Cytotoxicity Mediuma.
2. Add the anti-RT2 monoclonal antibody to a final concentration of 1:20 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 Cytotoxicity Mediuma with the appropriate concentration of Cedarlane
    Baby Rabbit Complement (CL3441)b (a final dilution of 1:1)
 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.
    Cytotoxic Index (C. I.) can be calculated as follows:
C.I. = 100 x % cyt (antibody + complement) - % cyt (complement alone)
                           100% - % cyt (complement alone)

SPECIFICATIONS:
Clone: CC02
Recipient: BALB/c mouse

Donor: Ab4/Ab4 rabbit

Immunizing cells: Nonadherent spleen cells from Ab4/Ab4 rabbits after passage through a Degalan bead column
coated with anti-Ab4 IgG.

Ig Class: Mouse IgG2b
Format: Ascitic fluid, frozen.

Results:
Antibody Titration by Cytotoxicity Analysis:
Cell Source: Rabbit Thymocytes
Cell Concentration: 1.1x106 cells/ml
Complement: Cedarlane Baby Rabbit Complement b (CL3441)
Complement Concentration: 1:1
Procedure: As described under Recommended Methods for Determining Percentage of RT2 Bearing Cells in a
Population.
C.I. = Cytotoxicity Index =
       100 x % cyt (antibody + complement) - % cyt (complement alone)
                   100% - % cyt (complement alone)


                                90
                                80
                                70
                                60
                     C.I. (%)




                                50
                                40
                                30
                                20
                                10
                                 0
                                     10

                                          20

                                               40

                                                    80

                                                         160

                                                               320

                                                                     640

                                                                           1280

                                                                                  2.5K

                                                                                         5K

                                                                                              10K

                                                                                                    20K

                                                                                                          40K




                                               Reciprocal of Antibody Dilution, k=1000
Tissue Distribution by Cytotoxicity Analysis:
Procedure: see page 3
Antibody Concentration Used: 1:12

Cell Source                                  C.I.
Thymus                                       80
Spleen                                       17
Bone Marrow                                   0
Peritoneal Exudate Cells                      9
Appendix:                                     0

Functional Testing:
Cell Source: Rabbit peripheral blood lymphocytes
Cell Concentration: 1 x 107 cells/ml.
Antibody Concentration: 1:6
Complement: Cedarlane Baby Rabbit Complement (CL3441)b at 1:2 dilution.

Results:
Rabbit lymphocytes were treated as described on page 2 “Recommended
Method for Depleteing a Rabbit Cell Population of RT2 Bearing Cells.”
Remaining viable lymphocytes were exposed to the mitogens concanavalin
A (CON A), phytohaemaglutinin (PHA) and lipopolysaccaride (LPS).

Cell depletion with RT2 antibody was found to have little effect on the LPS
response and to inhibit the CON A and PHA response. (87% & 80%
respectively).

NOTES: There may be variation in percent kill from rabbit to rabbit.

a.    Cedarlane Cytotoxicity Medium (CL95100) is RPMI-1640 with 25 mM 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 of complement. Some batches of BSA also contain complement-dependent
      cytotoxins, resulting in the same problem. We screen for batches of BSA giving low background in the
      presence of complement and use the selected BSA for preparing Cedarlane Cytotoxicity Medium.

b.    Baby rabbit serum is pooled from normal rabbits 3-4 weeks old. It is a potent source of complement and low in
      non-specific lymphocytotoxins.

REFERENCES:
1) Ponsard, D.C., C.T. Chou, B. Cinader, and S. Dubiski 1986. The
   rabbit immune system: Cells regulating the proliferative response of
   purified T cells to Concanavalin A and phytohaemagglutinin.
   Immun. Letter 13: 63-69.

2) Lieu, Y., C.T. Chou., and S. Dubiski, 1984. T cell antigens and
   MHC determinants on rabbit granulocytes. Immun. Letter 7: 181.

3) Ponsard, D.C., B. Cinader, C.T. Chou, and S. Dubiski, 1986.
   Characterization of rabbit cells by monoclonal and polyclonal
   antibodies. Immun. 59: 115-122.

4) Cinader, B., S. Dubiski, C.T. Chou, and L. Charpentier, 1988.
   Fluorescence-Activated Cell Sorter (FACS) Analysis of Rabbit Cells.
   Cellular Immunology. 112: 001-009.


                                        FOR RESEARCH USE ONLY
                           ® is a Registered Trademark of Cedarlane Laboratories Limited.


JB 2002/05/15

								
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