|REACTIVITY||H M R|
|Flow Cytometry (Fixed/Permeabilized)||1:50|
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised December 2010
Protocol Id: 220
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
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human calreticulin protein.
Calcium is a universal signaling molecule involved in many cellular functions, such as cell motility, metabolism, protein modification, protein folding, and apoptosis. Calcium is stored in the endoplasmic reticulum (ER), where it is buffered by calcium binding chaperones such as calnexin and calreticulin, and is released via the IP3 Receptor channel (1). Calreticulin also functions as an ER chaperone that ensures proper folding and quality control of newly synthesized glycoproteins. As such, calreticulin presumably does not alter protein folding but regulates proper timing for efficient folding and subunit assembly. Furthermore, calreticulin retains proteins in non-native conformation within the ER and targets them for degradation (2,3).
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