Immunoassays

IMMUNOASSAYS

complement fixation test

cytokine quantification

multiplex cytokine analysis

ELISpot

immunocytochemistry

immunohistochemistry

T-cell expansion

antigen-specific CD8+ T cells

immunohematology

HLA typing

lymphocyte microcytotoxicity assay

mixed lymphocyte culture

primed lymphocyte typing (PLT)

cytotoxic lympholysis

lymphocyte depletion studies

cytotoxicity assays

complement hemolytic activity (CH₅₀)
alternative pathway hemolytic activity (AH₅₀ / AP₅₀)
complement (deviation and) fixation test (CFT) detects IgM and IgG₃ >> IgG₁ > IgG₂ against a particular antigen.

complement fixation / fixation of complement / Bordet-Gengou phenomenon or reaction : the consumption of complement upon reaction with immune complexes containing complement-fixing antibodies, the basis of CFT

Protocol for CFT :

treatment of patient's serum :

spontaneous denaturation of complements factors is accelerated by heating it at 56 °C for 30': such treatment doesn't denature Igs
elimination of eventual cross-reactive Forssman Abs in patient's serum by absorption to sheep RBCs, which (on the contrary of human ones) have Forssman Ag .

add purified targeted antigen(s)
add fresh guinea pig (Cavia porcellus ) or rabbit (Oryctolagus cuniculus ) serum, containing a recently titred dose of complement factors (such small-sized species are preferred as they rarely have dyscomplementemias) and incubate at 37°C per 30'.
add sensitized sheep RBCs (SSRBC) as indicator system = sheep RBCs with adhesed anti-sheep RBCs antibodies (hemolysin / amboceptor) obtained from inoculation of sheep RBCs into rabbit.
if complement factors had bound eventual anti-antigen antibodies (deviation), they are consumed and poorly fix to anti-sheep RBCs antibodies on SSRBCs. So the latter don't undergo hemolysis, i.e., a positive test result. Otherwise complement-mediated SSRBCs hemolysis occurs and solution (first torbid) becomes clean (quantitative measure by spectrophotometry). Quantitative results are obtained by determining the highest dilution of antiserum or test material that gives a positive reaction.

An effectiveness control is created using only steps c) and d) : if all is right SSRBCs hemolysis occurs.

Presence of anti-complement factor(s) Abs in patient's serum is demonstrated by using only steps a), c) and d).

CH₅₀, hemolytic, total, or whole complement assay : a functional assay of total complement activity that measures the capacity of serial dilutions of serum to lyse a standard preparation of sheep red blood cells coated with antisheep erythrocyte antibody. The reciprocal of the dilution of serum that lyses 50 per cent of the erythrocytes is reported as the whole complement titer in CH50 units per milliliter of serum

CH₅₀ unit : the amount of complement that will lyse 50% of a standard preparation of sheep red blood cells coated with antisheep erythrocyte antibody.

amboceptor unit : in complement fixation tests, the smallest amount of anti-RBC antibody (amboceptor) that produces complete red cell lysis in the presence of an excess of complement.
antigen unit : in complement fixation tests, the smallest amount of antigen that will fix one unit of complement.
complement or hemolytic unit : in complement fixation tests, the smallest amount of complement or serum that will produce complete hemolysis of sensitized red cells
complement deviation / Neisser-Wechsberg phenomenon : inhibition of complement fixation or complement-mediated immune hemolysis in the presence of excess antibody

radioallergosorbent test (RAST)

Phadebas-RAST^®
Pharmacia CAP System^®
UniCAP System^®

cytokine quantification : cytokines can be quantitated at various levels.

mRNA can be detected by real-time PCR : multiplex assays for detection of cytokines at the mRNA level are commonly used^ref
intracellular proteins can be measured by FACS staining of permeabilized cells : multiplex assays for detection of cytokines are commonly used^ref1,
ref2. However, these assays have one or more limitations, like the need for a large sample volume or detection of precursor proteins rather than native secreted proteins. In addition, these techniques are time-consuming and laborious.
secreted cytokines can be quantified with ...

bioassays
enzyme-linked immunosorbent assays (ELISAs)
radioactive immunosorbent assays

multiplex cytokine analysis technologies have become readily available since 2001. They detect up to 100 different cytokines in the same volume used for an individual ELISA. 2 main formats exist:

multiplex sandwich ELISA

FAST Quant
SearchLight

bead based multiple immunoassays (MIA)

UpState Luminex
Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology^ref

cytokine secretion profiling

ELISpot (cytokine typization at the single cell level)

single cell suspensions are distributed in PVDF bottomed wells (usually 96) previously coated with an anti-cytokine antibody diluted in PBS and incubated overnight at 4°C. Plates are washed with PBS and then blocked for 2h at room temperature with PBS supplemented with 1% BSA and 5% sucrose. Cells are added in triplicates at an input cell number of 2 x 10⁵ cells per well resuspended in complete culture medium (RPMI supplemented with 10% FBS and antibiotics). These cells may upon stimulation with the proper antigen (added at a final concentration of 5 mg/ml) produce cytokine molecules in their close environment which are caught by the coated antibody. Concanavalin A at 5 mg/ml, and complete culture medium are used as positive and negative control, respectively. Plates are washed after a 24 h (for IFN-g) or 48 h (for IL-4) incubation at 37°C, in a 5% CO₂ atmosphere incubator.
after cell removal, the captured cytokine is revealed by a secondary biotinylated anti-cytokine antibody, which is in turn
detected by streptavidin conjugated to AlkP
the enzymatic reaction produces coloured spots that can be counted on an inverted microscope, allowing the numeration of cytokine producing cells under a given stimulus.
plates are air-dried and the spots are counted using a steromicroscope at a 40x magnification and an automated ELISpot reader system (CTL Analyzers, Cleveland, OH) with the ImmunoSpot software. The average number of spot forming cells (SFC) is adjusted to 1 x 10⁶ cells.

immunocytochemistry (ICC)

ABC technique for VIP : following perfusion with a picric acid-paraformaidehyde fixative, tissues are removed ' washed in sucrose-P04 buffer solution, and sectioned with a freezing microtome.. Tissue sections are processed for vasoactive intestinal polypeptide (VIP) immunohistochemistry using an adaptation by Jean Y. Jew, M.D., of the ABC method (Hsu, et al, J Histochem Cytochem 29: 577-588, 1981). Briefly, tissues were sequentially incubated in the following solutions, with washes in PO,-buffered saline between incubations: VIP antiserum, diluted 1:1000; biotinylated goat anti-rabbit IgG; avid in-bioti nylated peroxidase complex; and substrate-containing diaminobenzidine (DAB) and H201 in Tris buffer. After the DAB visualization step and wash in Tris buffer, sections are mounted on chrome alum gelatin-coated slides, dried, dehydrated through a graded ethanol and xylene series, and coverslipped with Permount

Web resources

Immunocytochemistry resources collection from Analytical imaging facility of Albert Einstein College of Medicine, Yeshiva University
Electron Microscopy Immunolabeling
Whole mount immunocytochemistry in Klymkowsky Lab Mannual
Immunofluorescent staining of mouse and rat leukocytes

surrogates :

analysis of serum IgG subclasses (i.e. analysis of T lymphocyte polarization). In mice :

T_h1 type response (IgG_2a)
T_h2 type response (IgG₁)

immunohistochemistry (IHC)

immunoperoxidase technique : a method of histologic staining in which a peroxidase-labeled antibody that binds to antigen is added to tissue, and the sites of its localization are revealed by addition of a chromogenic substrate system that produces a colored reaction product visible by light microscopy
peroxidase-antiperoxidase (PAP) technique : a technique for detecting antigen or antibody in tissue sections. The tissue section is incubated with rabbit antibody specific for the antigen to be detected, followed by an excess of antirabbit IgG. A complex of horseradish peroxidase and rabbit antiperoxidase is added; these are linked to the antigen-bound antibody by the antirabbit IgG. The PAP complexes are then stained by incubation with a chromogenic substrate to produce a colored reaction product

Web resources

IHC World
Immunoportal.com : Immunohistochemistry - In situ hybridization by Hogne Roed Nilsen
Immunohistochemistry Home Page by Mahmood Aijazi
Troubleshooting tips of IP, western and immunostaining from Chemicon
Usenet sci.bio.immunocytochem
Journals :

T-cell expansion

conventional T-cell expansion methods require that researchers maintain a separate line of antigen-presenting cells (APCs). You have to use live cells, and keep them in culture, and feed them.
paramagnetic particles : Oslo-based Dynal Biotech has built an entire company around its Dynabeads, which are tiny paramagnetic particles that bind target cells, proteins, or nucleic acids, and can be removed in seconds with a magnet once their work is done. Dynal's newest kit, the Mouse CD3/CD28 T-cell Expander, eliminates much of the tedium from the task of T-cell expansion. Released to complement Dynal's human T-cell expander, as well as its recently launched kit for mouse B-cell negative isolation, the kit bridges the gap between human research and animal models. The cell-sized Dynabeads (4.5 mm in diameter) are coated with anti-CD3 and anti-CD28 monoclonal antibodies to mimic the effects of APCs, which stimulates the cultured T cells to proliferate. Once expansion is completed, the beads can be removed with a magnet, leaving the T cells behind. It's a much cleaner process, since it doesn't introduce live cells that will later have to be removed

quantitative analysis of antigen-specific CD8⁺ T lymphocytes :

surface staining with MHC I-peptide tetramers
staining of peptide re-stimulated CD8⁺ T cells for intracellular IFN-g
⁵¹Cr-release assays
staining of antigen-activated lymphocytes (SAAL) : lymphocytes from mice vaccinated with TAAs were expanded for 5 days in tissue culture and then stimulated in vitro for 5 h with tumor cells. They were subsequently surface-stained for CD8 and for intracellular IFN-g and analyzed by flow cytometry. The specificity and sensitivity of this assay was comparable to that of . The assay did not exhibit the high background activity of traditional ⁵¹Cr-release assays that without elaborate effector cell purifications commonly fail to distinguish between T cell-mediated antigen-specific cytolysis and non-specific lysis by LAK cells. The described method, which does not require prior identification of individual TAAs and their T cell epitopes nor access to specific reagents such as MHC-peptide tetramers, represents a simple yet useful technique for studying tumor-specific cytolytic T cell responses^ref.

quantitative analysis of antigen-specific CD4⁺ T lymphocytes : most MHC class II tetramers are produced with a homogeneously built-in peptide, reducing greatly their flexibility of use. The generation of "empty" functional HLA-DR*1101 tetramers, receptive for loading with synthetic peptides by incubation, has been recently achieved. No such reagent is in fact available for this HLA-DR allele, one of the most frequent in the Caucasian population. Soluble MHC class II-immunoglobulin fusion proteins (HLA-DR*1101-Ig) were compared with soluble MHC class II protein fused with an optimised Bir site for enzymatic biotynilation (HLA-DR*1101-Bir), both produced in insect cells. The molecules were multimerised by binding fluorochrome-protein A or fluorochrome-streptavidin, respectively. HLA-DR*1101-Bir molecules are superior to the HLA-DR*1101-Ig ones both in biochemical and functional terms. HLA-DR*1101-Bir molecules can be pulsed with at least three different promiscuous peptide epitopes, derived from Tetanus Toxoid, influenza HA and the tumour associated antigen MAGE-3 respectively, to stain specific CD4⁺ T cells. Both staining temperature and activation state of CD4+ T cells are critical for the binding of peptide-pulsed HLA-DR*1101-Bir to the cognate TCR. It is therefore possible to generate a soluble recombinant HLA-DR*1101 backbone that is receptive for loading with different peptides to stain specific CD4⁺ T cells. As shown for other HLA-DR alleles, we confirm that not all the strategies to produce soluble HLA-DR*1101 multimers are equivalent^ref.
immunohematology

crossmatch / cross match / cross-match : a test of the compatibility of donor and recipient blood performed before transfusion: red cells of the donor are placed in serum of the recipient (major crossmatch) and red cells of the recipient in serum of the donor (minor crossmatch) and antiglobulin is added to increase reactivity; the presence of hemolysis or agglutination indicates incompatibility.
typing of blood groups : classification of the blood with reference to various erythrocytic membrane antigens
pretransplant or HLA crossmatch / cross match / cross-match : a test for the presence in the serum of a prospective transplant recipient of cytotoxic antibodies against donor tissue antigens: donor lymphocytes are placed in serum of the recipient; the presence of cytolysis indicates incompatibility and the likelihood of hyperacute graft rejection
MHC, HLA or tissue typing : determination of the HLA antigens possessed by an individual. HLA typing is used to identify compatible donors and recipients for transplantation or platelet or granulocyte transfusion, to establish associations of HLA antigens with diseases, and in paternity testing.

PCR of MHC locus
class I antigens (HLA-A, -B, and -C) are detected by lymphocyte microcytotoxicity assay : multiple standard typing sera are placed in wells of a microtiter plate, and peripheral blood lymphocytes and complement are added to each well. The pattern of lysed cells indicates the HLA phenotype.
class II antigens are detected by lymphocyte proliferation assay or test / blastogenesis assay : a functional test of the ability of lymphocytes to respond to mitogens, specific antigens, or allogenic cells. Lymphocytes are cultured both with and without the stimulant for several days and then are cultured for 16 hours with 1 mCi ³H-thymidine per well. ³H-thymidine incorporation is measured in a Wallac b-counter. The means counts per minute is calculated for triplicates of antigen, mitogen or the control medium. The ratio of the the mean counts per minute in the stimulated (by antigen or mitogen) and control cultures is reported as the stimulation index (SI) or ratio (SR). An SI > 3 is defined as positive. All 3 types of stimulants are used in investigation of immunodeficiency. Commonly used mitogens are phytohemagglutinin (PHA), concanavalin A (ConA), and pokeweed mitogen (PWM); commonly used antigens are PPD (tuberculin), Candida antigen, and streptokinase-streptodornase

mixed lymphocitary reaction (MLR) / mixed lymphocyte culture (MLC) : the lymphocyte proliferation assay with allogenic cells, commonly performed for transplantation tissue typing, in which lymphocytes from 2 individuals are cultured together and the proliferative response (mixed lymphocyte reaction) is measured by ³H-labeled thymidine uptake

autologous MLR (AMLR) : T cells cultured in vitro with autologous APC undergo low level proliferation that can generate effector cells that either suppress polyclonal Ab production or mediate autocytotoxic activity. This AMLR is MHC class II restricted and the antigenic peptides involved are mainly cleaved fragments of self-Ags produced through the process of apoptosis .
heterologous MLR

two-way heterologous MLR : lymphocytes from 2 different individuals (or different mouse strains) are mixed and incubated in culture for 5 days, after which ³H-Thy is added to the culture. 5 hours later the cells are harvested, and if they recognize foreign antigens, they will have incorporated ³H-Thy into their DNA because they have divided (mounted an immune response).
one-way heterologous MLR : 1 of the 2 cell populations is treated with inhibitors of cell division (radiations or mitomycin C ), so that one can show cell division in the other cell population.

homozygous typing cells (HTC) : cells homozygous for a known HLA-D specificity; panels of HTC of all established HLA-D types are used to determine the HLA-D type of unknown cells using one-way mixed lymphocyte reactions.

early or primary MLR : mainly depends on HLA-DR (> HLA-DQ) alleles

late or secondary MLR (after 24÷36 hrs) : mainly depends on HLA-DP alleles

primed lymphocyte typing (PLT) : a technique used for typing of class II HLA antigens: unknown cells are exposed to a panel of lymphocytes primed against specific HLA antigens by prior coculture with stimulator cells that matched the primed cells at all but one HLA locus; when restimulated by the same HLA antigen the primed cells give a secondary proliferative response, which shows that the unknown cells bear the same antigen as the stimulator cells
cytotoxic lympholysis (CTL) / cell-mediated lympholysis (CML) : a variation of the mixed lymphocyte culture (MLC) technique that is a functional test of the ability of cytotoxic lymphocytes (CTL) to kill target cells. Lymphocytes from 2 different individuals (or different mouse strains), one of which has been prevented from proliferating by treatment with radiation or mitomycin C (a “one-way” MLC), are mixed and incubated for 5 days, after which ⁵¹Cr-labeled target cells (cells with the same surface antigens as the cells that were treated with mitomycin C) are added to the culture. 5 hours later, the supernatant of the culture is recovered and will be radioactive (compared to percentage of ⁵¹Cr released from control (nonspecific target) cells) if the target cells have been attacked and lysed (the ⁵¹Cr has been released) by the cells that were not treated with mitomycin C. This response is a CMI.
DR Class II antigens are also detected by lymphocyte microcytotoxicity assay using B-lymphocytes and anti-DR antibody types

Web resources

South Thames Tissue Typing
Tissue typing by Gareth Page and Paul Norman

lymphocyte depletion studies :

CD8 depletion is performed by intraperitoneal injection of anti-CD8 antibody 2.43 at 0.5 mg/mouse, starting 1 day before the first vaccination (priming phase) or 3 days after the last vaccination (effector phase) and repeated weekly
CD4 depletions is performed by intraperitoneal injection of anti-CD4 antibody GK1.5 at 0.5 mg/mouse, starting 1 day before the first vaccination (priming phase) or 3 days after the last vaccination (effector phase) and repeated weekly
NK cell depletions were achieved by intraperitoneal injection of anti-asialo GM₁ antibody (Wako Chemicals USA, Richmond, VA) at 20 µL per mouse, starting 1 day before or 3 days after vaccination and repeated every 5 days

cytotoxicity assays :

NK-dependent cytotoxicity is measured on the MHC class I- and II-negative human myelogenous leukemia cell line, K562
inhibitors of granule-dependent cytotoxicity :

concanamycin A, an inhibitor of vacuolar type H+-ATPase, 10 nM for 2 h, selectively block the perforin and granzyme cytotoxicity pathway, mostly by accelerated degradation of perforin caused by an increase in the pH of lytic granules^ref1,
ref2
ethylene-bis(oxyethylenenitrilo)-tetraacetic acid (EGTA), a chelator of calcium, 4 mM for 12 h
strontium chloride 25 mM for 18 h^ref1,
ref2 induces degranulation of mast cells and T lymphocytes^ref1,
ref2 without causing cellular toxicity and is used to deplete granules^ref1,
ref2

viability of cells is assessed by measuring :

molecules released from dead cells :

radioisotopes :

⁵¹Cr release assays (Brunner, 1968^ref)^ref: briefly, 1 x 10⁴ ⁵¹Cr (Na₂⁵¹CrO₄; New England Nuclear, Boston, MA)-labeled target cells and various numbers of effector cells (effector to target (E/T) cell ratios of 50:1, 25:1, 5:1, and 1:1) in 200 µL of RPMI 1640 supplemented with 10% FCS were seeded into round-bottomed microtiter wells and incubated for 4 hours^ref1,
ref2. In some experiments, the target cells were incubated with an anti-HLA class I framework MoAb (w6/32; American Type Culture Collection, Manassas, VA) or an anti–HLA-DR MoAb (L243; American Type Culture Collection) at an optimal concentration (10 µg/mL) for 30 minutes before adding effector cells to determine whether cytotoxicity was restricted by HLA class I. To determine whether WT1-specific CTLs lyse myeloma cells via recognition of the WT1 peptide, which is naturally processed in myeloma cells and expressed in the context of HLA-A24, cold target inhibition assay was performed. WT1 peptide-loaded and unloaded autologous LCL or HLA-A24–positive leukemia cell line MEG01, which was shown to be lysed by WT1-specific CTLs in a WT1-specific manner, was used as cold target cells. After incubation for 4 hours, 100 µL of supernatant were collected from each well. The percentage of specific lysis was calculated as follows: (experimental release cpm – spontaneous release cpm)/(maximal release cpm – spontaneous release cpm). Cytotoxicity mediated by purified perforin was measured by using 2-hour ⁵¹Cr release assays and the trypan blue exclusion method. ⁵¹Cr-labeled target cells were incubated with various concentrations of purified perforin in the assay buffer [150 mmol/L NaCl, 20 mmol/L HEPES, and 2.5 mmol/L CaCl₂ (pH 7.4)] for 2 hours at 37°C. After incubation, supernatants were harvested after centrifugation of the microtiter plates, and radioactivity was determined. The major disadvantage results from the use of a radioactive compound, with the related problems of handling and disposal due to the short half-life of the isotope. The results obtained by the micro-cytotoxicity assay (500 target cells) were comparable to those of the standard assay (5000 target cells) and ⁵¹Cr release evaluation using the gamma counter was the most sensitive method of determining lytic activity using 500 tumour target cells. beta counter evaluation using solid phase scintillation was found to be a reproducible alternative method, even if the lytic curves cannot be compared with those obtained using the traditional method^ref.

reporter enzymes released in the supernatant by dead targets (convenient, inexpensive and precise)

transfected

Escherichia coli b-galactosidase release (BGR) (after transfection with vaccinia virus^ref or plasmid^ref) evaluated using

the chemiluminescent substrate, AMPGD (3-4-methoxyspiro[1,2-dioxetane-3, 2'-tricyclo(3.3.1.1(3,7))decan]-yl--markphenyl-b-D-galactopyranoside), a phenylgalactose-substituted 1,2-dioxetane compound^ref
4-methylumbelliferyl-b-D-galactoside, a fluorescent substrate^ref

endogenous

lactate dehydrogenase (LDH)^ref : spontaneous release considerably lower than for ⁵¹Cr
alkaline phosphatase^ref

variations in metabolic activity on nonradioactive compounds, which is directly proportional to the number of viable cells

tetrazolium salt reduction (MTT) assay^ref
dimethylthiazol-diphenyl bromide tetrazolium bromide
methylumbelliferyl heptanoate
Alamar blue : is a non-toxic metabolic indicator of viable cells that becomes fluorescent upon mitochondrial reduction. Since alamarBlue reagent is non-toxic to cells and the assay can be performed under sterile conditions, effector cells may be recovered at the end for further analysis or cell expansion, if desired^ref

fluorochromes

used for prelabeling target cells measure the amount of dye released from or remaining in prelabeled target cells. The major drawbacks for these methods appear to be the high spontaneous release of the fluorescent dye, the slow specific release of the fluorescein dye, and the low intensity of the fluorescence signal, all of which decrease the overall sensitivity of the test

dyes retained by living cells :
dyes released by died cells :

release of the activated fluorochrome calcein AM from lysed cells^ref
calcein-acetoxymethyl (calcein-AM) release assay (CaRe-LAss)^ref : calcein-AM, a non-fluorescent lipid-soluble diester which passively crosses the cell membrane and is intracellularly converted to a polar lipid-insoluble fluorescent product (calcein) by esterase activity in viable cells, is initially used to stain target cells. In cells with intact plasma membranes, displays good retention characteristics and low pH sensitivity^ref, and there is no stain transfer among cells^ref. After incubating targets with effectors for 2 h, ethidium homodimer-1 (EtD-1), a red DNA stain non-permeable to viable cells, is added. Dead target cells are distinguished by their double (green-red) staining. Data analysis is performed on FACS by gating the regions of living target, dead target and living effector cells, based on appropriate controls (excited at 530 and 645 nm, and emissions acquired at 530 and 645 nm, respectively). Non-specific events are subtracted from the dead target region and the ratio of specific dead target events to total target events is expressed as percent cytotoxicity^ref1,
ref2. Release of calcein in the supernatants recovered from cytotoxicity assays can be measured rapidly and with a high level of sensitivity by a fluorimeter. Alternatively, lytic activity could be determined by measuring the fluorescence retained in living cells after quenching the fluorescence released by dead targets^ref1,
ref2. Frequently, cytotoxicity tests are limited by the number of available effector cells, as for poorly represented cellular subpopulations (such as NK subsets and sorted or cloned lymphocytes) or by small amounts of blood (e.g., from children and elderly people). In these situations, the ability to use a low number of effector cells and the ability to recover cells after the cytolytic activity test would greatly facilitate the performance of the assays. The intense green fluorescent stain is stable for at least 4 h^ref. Calcein-AM cytotoxicity assays based on the evaluation of dye released in the supernatant used a relatively high number of target cells (15 × 10³)^ref; the number of targets used for the evaluation of dye retained by living cells (3 × 10³) was also relatively high^ref, except in a study in which the test was performed using Terasaki trays and 5 × 10² targets in 5 µl of medium^ref. The method requires very small quantities of cells to evaluate the dye released in the supernatant (500) and maintains the same experimental procedures and working volumes as in the⁵¹Cr assay. A strict correlation between results was found, with no significant differences in the experimental release values, making it possible to compare data obtained using the 2 different techniques. Furthermore, by reducing in the number of cytolytic effector and target cells 10-fold, without reducing working volumes, we obtained results very similar to those of the standard calcein-AM test. The microassay is therefore as reliable as the standard one, and this is of great importance when small amounts of blood are available, as in studies on elderly people or children. Fluorescent calcein released from lysed cells can be easily and readily measured in the recovered supernatants using a standard automated plate-reading fluorimeter. The entire procedure is analogous to the chromium release assay but requires less time to perform. In fact, while the standard ⁵¹Cr assay requires 1 h to label target cells, calcein-AM labeling is performed in 30 min. In addition, the former method has slow processing times when large numbers of samples have to be counted (1 min per well), while with the calcein-AM assay and the Spectramax fluorimeter the detection time can be standardized to less than 1 s per well. Spontaneous release of calcein is significantly higher than that of ⁵¹Cr, particularly in the microassay, in accordance with previous findings^ref. This higher spontaneous release does not depend on poor target cell viability caused by calcein-AM, as demonstrated by performing a double labeling of K562 cells with calcein-AM and subsequently with 51Cr. The technique shows sensitivities similar to those for ⁵¹Cr labeling while maintaining specificity. In effect, the difference between results never reached statistical significance, both assays revealing the same functional correlation. The slightly higher sensitivity of the calcein-AM assay could allow a reduction of the incubation time to less than 4 h, probably even reducing the spontaneous release rate. However, spontaneous release did not seem to be influenced by the dye concentration^ref. ^ref
4-methylumbelliferyl heptanoate (MUH) : the assay is based on the hydrolysis of the fluorochrome (MUH) by intracellular esterases of viable cells resulting in the production of highly fluorescent 4-methylumbelliferone that can be measured in a microplate fluorimeter. Because of a similarity to the principle of the widely used colorimetric MTT assay, a comparison was made between the two assays when measuring cell proliferation and LAK cell cytotoxicity to different target cell types. The MUH assay represents a method for evaluating both cell-mediated cytotoxicity and cell proliferation which is completely comparable to the MTT method. The rapidity of the new cytotoxicity assay, 5 h in contrast to 9 h for the MTT assay, its applicability to both adherently and nonadherently growing target cells and its high accuracy due to the avoidance of centrifugation steps make this method a serious contender for replacing conventional radioactive techniques^ref
fluorescent lanthanide chelates^ref : europium (Eu³⁺)^ref1,
ref2 or terbium
PKH-2, c'FDA^ref
D275 (a green fluorescent membrane dye) used to label various target cell lines and propidium iodide (PI) uptake (a nuclear dye) was used to assay cell death^ref
rhodamine-123^ref
carboxyfluorescein diacetate (CFDA) : both sensitivity and specificity of the presented method were higher than the ⁵¹Cr release assay. Moreover, the detection of human NK activity against K562 target cells required only 2 hrs, compared to 4 hrs in the standard ⁵¹Cr release assay^ref
2',7'-bis-carboxyethyl-carboxyfluorescein (BCECF)^ref1,
ref2

excluded by living cells :

trypan blue exclusion
7-amino actinomycin D (7-AAD) exclusion

cell growth curves

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