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CD CLUSTERED ANTIBODIES  

(anti-Human and others as indicated)

RDI Divison of researchd Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (flow cytometry, neutralization, blotting, histochemistry, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below. All products are for in vitro research use only-not for use in or on humans or animals-not for use in diagnostics. Price/availability/specifications subject to change without notice.


CD44 Antibodies


Mouse anti-human CD44 antibodies

-3 clones 


CATALOG#                 CLONE#                  Workshop    Host         Form        Price

RDI-CBL154                 F10-44-2                        V            mIgG2a     purified     $375.00

RDI-CBL154FT                " "                                 "               "             FITC        $375.00

RDI-CBL154PE                " "                                 "               "             PE            $469.00

RDI-M1676clb               P2                                   --           mIgG1       purified     $438.00

RDI-M1678clb                  " "                                --               "            FITC        $438.00

RDI-CD44-Hermes1      HERMES-1                   --            ratIgG2a    purified     $562.00 (multi-species reactive)


Product Specification: mouse monoclonal anti-human CD44

PeliCluster  CD44

Cat#RDI- M1676clb  $438.00/vial

Test/vial 200

Clone NKI-P2

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with immunoprecipitated LFA-I family antigen. This antibody has been clustered to CD44 in one of the international Workshop on Human White Cell differentiation Antigens

Isotype Mouse IgG1.

Source Mouse ascitic fluid.

Purification Ammoniumsulphate precipitation followed by ionexchange chromatography.

Packing Each vial contains 1 ml with approximately 0.2 mg/ml monoclonal antibody and 10 mg BSA in 20 mM TRIS and 150 mM NaCl, pH 8.0.

Preservative Mertiolate (0.001%).

Storage and stability Monoclonal antibodies should be stored in the dark at 2-8°C. The reagent is stable until the expiry date stated on the vial label.

Major reactivity The monoclonal antibody is directed against the CD44- antigen, which is mainly expressed on leucocytes, erythrocytes and weakly on platelets. The CD44-antigen is a transmembranous molecule which has undergone both extensive O-linked and N-linked glycosylation.

Molecular mass 80-95 kDa.

Application Identification of CD44 positive cells in immunocytochemistry and flow cytometry.

Studies involving T-cell, B-cell, monocyte and granulocyte binding to HEV during lymphocyte circulation and movement of leucocytes to inflammatory sites.

Studies on HCAM (homing receptor) function.

Methods Indirect immunofluorescence staining with analysis by flowcytometry or fluorescence microscopy.


References 1. Goldstein, L.A.& Butcher, E.C., Immunogenetics, 32, (6), 389-97 (1990).


APPLICATION  FOR AN INDIRECT IMMUNOFLUORESCENCE TECHNIQUE

Reagents

- FITC-conjugated goat anti-mouse immunoglobulin antiserum (M1204).

    Remove aggregates by centrifugation at 1000 x g for 10 minutes.

- Buffer: Phosphate Buffered Saline (PBS), containing 0.2% Bovine Serum Albumin (BSA).

- PFA: Para-Formaldehyde 1%, pH 7.2, containing 0.2% BSA.

- Microtiter plates (96 wells, V bottom) or tubes.


Procedure

1 Fixate the cells with PFA 1% and wash them with buffer.

2 Prepare a mononuclear cell suspension with a concentration of 1 x 10 7 cells/ml in buffer.

3 Add 45 µl of cell suspension to the microtiter wells or tubes.

4 Add 5 µl monoclonal antibody to the microtiter wells or tubes and mix gently.

5 Incubate for 30 minutes at 2-8 C.

6 Add 100 µl buffer to the microtiter wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

7 Aspirate the supernatant from the cell pellet and resuspend the cells.

8 Add 200 µl buffer to the microtiter wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

9 Aspirate the supernatant from the cell pellet and resuspend the cells.

10 Add 50 µl FITC-conjugated goat anti-mouse immunoglobulin serum, solid phase adsorbed with human immunoglobulins,     diluted 1:80.

11 Incubate for 30 minutes at 2-8 C.

12 Repeat step 6-9.

13 Add 200 µl buffer.

14 The number of positive cells can be determined by flowcytometry analysis or by means of fluorescence microscopy.


NOTE: Care should be taken when drawing blood to avoid activation of platelets. Platelets activation markers are weakly expressed on platelets prepared from directly fixed normal whole blood.

The expression is maximal on thrombin (1 U/ml) stimulated washed platelets.


FOR IN VITRO RESEARCH USE ONLY


Product Specification: mouse monoclonal CD44,conjugated to   Fluorescein
PeliCluster  CD44 F


CAT#RDI-M1678clb

Test/vial 100

Clone NKI-P2

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with immunoprecipitated LFA-I family antigen.

This antibody has been clustered to CD44 in one of the international Workshop on Human White Cell differentiation Antigens

Isotype Mouse IgG1.

Source Mouse ascitic fluid.

Purification Ammoniumsulphate precipitation followed by ionexchange chromatography.

Conjugation Conjugated with fluorescein iso thiocyanate isomer 1 (FITC).

Molecular F/P ratio between 5.5 - 9.5.

Packing Each vial contains 1 ml FITC conjugated monoclonal antibody and 10 mg BSA in PBS.

Preservative Mertiolate (0.001%).

Storage and stability Monoclonal antibodies should be stored in the dark at 2-8°C. The reagent is stable until the expiry date stated on the vial label.

Major reactivity The monoclonal antibody is directed against the CD44- antigen, which is mainly expressed on leucocytes, erythrocytes and weakly on platelets. The CD44-antigen is a transmembranous molecule which has undergone both extensive O-linked and N-linked glycosylation.

Molecular mass 80-95 kDa.

Application Identification of CD44 positive cells in immunocytochemistry and flow cytometry.

Methods Direct immunofluorescence staining with analysis by flowcytometry or fluorescence microscopy.

References 1. Goldstein, L.A.& Butcher, E.C., Immunogenetics, 32, (6), 389-97 (1990).

APPLICATION in direct Immunofluorescence techniques

Method with ficoll purified cells


Reagents and materials

- FITC-conjugated monoclonal antibodies.

- Buffer: Phosphate Buffered Saline (PBS), containing 0.2% Bovine Serum Albumine (BSA)

- PFA: Para-Formaldehyde 1%, pH 7.2, containing 0.2% BSA

- Microtiter plates (96 wells, V bottom) or tybes.


Procedure

1 Prepare a mononuclear cell suspension with a concentratation of 1 x 10 7 cells/ml.

2 Ad 40 µl of cell suspension to microtiter wells or tubes.

3 Add 10 µl of the antibody to the microtiter wells or tubes and mix gently.

4 Incubate for 30 minutes at 2-8°C.

5 Add 150 µl buffer to the microtiter wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

6 Aspirate the supernatant from the cell pellet and resuspend the cells.

7 Add 200 µl buffer to the microtiter wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

8 Aspirate the supernatant from the cell pellet and resuspend the cells.

9 Cytoflow fluorometer analysis:

    Add 200 µl buffer to the microtiter wells and transfer this final cell suspension to appropriate testtubes, or add 200 µl             buffer to the tubes.

10 If analysis within 8 hours is not possible add at no. 9, instead of buffer, 200 µl PFA 1%.


Fig. 1

Fluorescense profile of ficoll purified normal cells

(Scatter gates set on the lymphocyte fraction)


WHOLE  blood method

Due to sophisticated laser flowcytometry we are now able to distinguish populations of human lymphocytes, monocytes and granulocytes by their characteristics in different light scattering. By gating on the appropriate cell cluster (Fig.2) we can easily determine mononuclear cells, which have been immunofluorescently stained with monoclonal antibodies, and enumerate them.


Reagents and materials

- FITC-conjugated monoclonal antibodies.

- Lysing solution (NH 4Cl, pH 7.2).

- Buffer: PBS, containing 0.2 % BSA.

- PFA: Para-Formaldehyde 1%, pH 7.2, containing 0.2 % BSA.

- Tubes.


Procedure

1 Draw blood into a blood collection tube containing EDTA.

2 Deliver 100 µl (*) of well mixed whole blood to the bottom of the testtube.

3 Add 10 µl of the undiluted antibody to the bottom of the testtube, and mix gently.

4 Incubate for 30 minutes at 2-8°C or in an ice-water bath.

5 Mix the tubes and add 2 ml of lysing solution.

6 Incubate for 3-5 minutes at room temperature.

7 When lysing is completed, centrifuge the tubes at 500 x g for 5 minutes.

8 Aspirate the supernatant from the cell pellet, resuspend the cells in 1 ml buffer.

   Place the tubes in the ice-water bath.

9 Analyse the samples within 8 hours.

If analysis within 8 hours is not possible centrifuge the tubes at 500 x g. Aspirate the supernatant from the cell pellet and resuspend the cells in 1 ml PFA 1%.


Fig. 2:

Scatter patern of a whole normal blood sample

R2 - lymphocytes R3 - monocytes R4 - granulocytes

Note:Use our special negative control to determine background fluorescence produced due to Fc binding capacities by mononuclear cells.

The concentration and F/P ratio of our control has been adjusted to our FITC-conjugated monoclonal antibodies. * This method was developed for blood samples with a normal white count. It may be necessary to adjust the quantity of blood for samples with very high or low white counts M1678/02 0708961329

FOR IN VITRO RESEARCH USE ONLY


MOUSE ANTI-HUMAN CD44 ANTIGEN (HCAM)


PRODUCT CODE :RDI-CBL154    $375.00/vial 200ug purified

FITC label 100 tests $375.00 ord cat#RDI-CBL154FT

    PE label 100 tests $469.00 ord cat#RDI-CBL154PE


CLONE: F10-44-2


ISOTYPE: IgG2a

Immunizing Antigen: purified human T cells Fusion Partner: NS1 myeloma cell line

SOURCE: tissue culture supernatant

PURIFICATION METHOD: Protein A affinity chromatography

SPECIFICITY: The mature CD44 antigen is a transmembranous molecule which has undergone both extensive O-linked and N-linked glycosylation. The main cellular reactivity is on leucocytes and red blood cells with only weak reactivity on platelets

APPLICATIONS: * Identification of CD44 - positive cells in immunocytochemistry and flow cytometry.

-formalin fixed paraffin wax embedded sections of lymphoid and epithelial tissues


* Studies involving T -, B - cell, monocyte and granulocyte binding to HEV during lymphocyte circulation and movement of leucocytes to inflammatory sites.

* Studies on HCAM (homing receptor) function.

-immunoprecipitation

REFERENCES: Dalchau, R. in Leucocyte Typing III. Oxford University Press 1987.

                          Dalchau, R. in Leucocyte Typing IV. Oxford University Press 1989.

                          Goldstein, L.A. & Butcher, E.C. Immunogenetics 32 (6) 389-97 (1990).

                          Stauder, R & Gunthert V. Immunologist 3, 78-83 (1995)

                          Favaloro, E. immunol Cell Biol. 71, 571-81 (1993)

                          McKenzie et al. J. Neuro Chemistry (1982)

                          Leucocyte Typing V, oxford Univ Press (1995)


PRESENTATION: Each monoclonal is delivered in a sealed vial at a concentration of not less than 200µg/2ml in phosphate buffered saline containing 10mM sodium azide and 1mg/ml of bovine serum albumin. We recommend that each laboratory determine an optimum working titre for use in its particular application.

STORAGE: Purified monoclonal antibodies are generally stable for long periods at 4oC. However repeated warming to room temperature and re-cooling may result in a loss of activity and hence effective working titre. It is recommended that monoclonal antibodies are aliquotted upon receipt and then stored at -20C. After thawing they should then be used as a stock solution for up to 60 days. Dilute working solutions are best prepared on the day of use.


For research use only. Not supplied for use in human diagnostic or therapeutic procedures


FOR BULK (NO BSA AND NO AZIDE): $1062.00/1 milligram


DATA SHEET: RAT anti-human CD44

Catalog#: RDI-CD44-Hermes1    $562.00/vial

LOT# see batch sheet

Package Size: 200ug purified antibody in 0.2ml PBS (no carrier or preservative).

Species: rat IgG2A

CLONE: Hermes-1

Activity: This monoclonal recognizes the human 80-95 kD lymphocyte surface glycoprotein that is involved in endothelial cell recognition and lymphocyte trafficking. Also recognizes CD44 homologues on rabbit, cow, goat and sheep lymphocytes1.

Application: Indirect immunofluorescence cell surface staining of leukocytes, most erythrocytes, epithelial cells and a wide variety of other tissue types.

-immunoprecipitation

-western blot

-immunohistochemistry

-flow cytometry

References: 1) de los Toyos, et al., 1989. Blood. 74(2):751

                    2) J Immunology March 15, 1989,142:2046- 2051

                    3) J Cell Bio 109:927-937

                    4) J Cell Bio Dec 1990, 111,2765-2774

Storage: Store at 4 DEG C for up to 30 days. Prolonged storage at -20 DEG C. Avoid repeated freeze thaw cycles.

Precautions: For In vitro research Use Only. Not   for use in or on humans or animals or   for diagnostics.


RDI Div of researchd Industries Intl

(Research Diagnostics Inc)

San Jose, 95123 CA Snell ave 658

USA

or 408-780-0908

email: sales@researchd.com

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