Cat. # | Size | Qty. | Price |
---|---|---|---|
72177S | 500 µl (100 tests) |
|
$ 216 |
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | |
Source/Isotype | Mouse IgG2b kappa |
Product Information
Application | Dilution |
---|---|
Flow Cytometry (Fixed/Permeabilized) | 1:20 |
Flow Cytometry (Live) | 1:20 |
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Count cells using a hemocytometer or alternative method.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to immunostaining.
NOTE: Human Fc receptors cross-react with rabbit IgG. When cells of interest express high levels of Fc receptor protein (for example, macrophage/monocyte lineages), pre-incubate live cells with human Fc block prior to immunostaining with rabbit antibodies.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
posted June 2017
revised January 2022
Protocol Id: 1504
Human
This monoclonal antibody was purified from tissue culture supernatant via affinity chromatography. The purified antibody was conjugated under optimal conditions, with unreacted dye removed from the preparation.
The protein phosphatase (PTP) receptor CD45 is a type I transmembrane protein comprised of a pair of intracellular tyrosine phosphatase domains and a variable extracellular domain generated by alternative splicing (1). The catalytic activity of CD45 is a function of the first phosphatase domain (D1) while the second phosphatase domain (D2) may interact with and stabilize the first domain, or recruit/bind substrates (2,3). CD45 interacts directly with antigen receptor complex proteins or activates Src family kinases involved in the regulation of T- and B-cell antigen receptor signaling (1). Specifically, CD45 dephosphorylates Src-family kinases Lck and Fyn at their conserved negative regulatory carboxy-terminal tyrosine residues and upregulates kinase activity. Conversely, studies indicate that CD45 can also inhibit Lck and Fyn by dephosphorylating their positive regulatory autophosphorylation site. CD45 appears to be both a positive and a negative regulator that conducts signals depending on specific stimuli and cell type (1). Human leukocytes including lymphocytes, eosinophils, monocytes, basophils, and neutrophils express CD45, while erythrocytes and platelets are negative for CD45 expression (4).
Several isoforms of CD45 are generated through alternative splicing in a cell type-specific and activation state-specific manner. The HI100 antibody is widely used as a leukocyte marker for naïve and activated T cells. Naïve T cells are positive for CD45RA, where activated T cells are negative for CD45RA (5).
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