Identification of Novel Sperm Surface Markers via HTS - Background

omarys's version from 2017-09-21 18:21

Section 0

Question Answer
Sperm may be washed by ___ ___ centrifugation or by a "direct swim-up" technique that does not involve centrifugation. (In normal semen samples, centrifugation causes no more DNA damage to spermatozoa than a direct swim-up technique.)density gradient
Sperm washing involves removing the seminal plasma and any cryoprotectant, if used, from the specimen. Done prior to ___.IUI
3 sperm washing techniquesBasic Sperm Wash, Premium Wash, Swim-up Technique

Section 1: Introduction

Question Answer
The human cell surface marker screening panel isa collection of cell surface marker antibodies
The human cell surface marker screening panel is designed toscreen cells for the expression of 373 human cell surface proteins
The human cell surface marker screening panel employs which biotechnology?flow cytometry
The significance of these panels is their potential for developing new strategies tocharacterize and isolate sperm from ejaculate of azoospermic patients (or from testicular biopsy)
There are ___ protein expression profiles at ___ states of differentiation.different ; various
HTShigh-throughput screening (and HTFC is flow cytometry)
The parameters measured in flow cytometry are- (3)fluorescence intensity (up to 18 colors); forward scatter; side scatter

Section 2: What's flow cytometry, precious? pt. 1

Question Answer
FC is a ___-based technology.laser
Applications are- (4)cell counting; cell sorting; detection of biomarkers; protein engineering
FCcharacterizes and defines different cell types in a heterogeneous cell population
FC also analyzes cell ___ and ___.size ; volume
FC is used to measure ___ ___ produced by fluorescent-labeled ___ detecting proteins, or ___ that bind to specific cell-associated molecules.fluorescence intensity ; antibodies ; ligands
In the cytometer, cells/particles passing through the laser beam ___ light, which is detected as ___ and ___.scatter ; forward scatter ; side scatter
FS correlates with ___ ___ and SS is proportional to the ___ of the cellscell size ; granularity (so using FS and SS alone helps differentiate cell size and granularity)
When running a blood sample thru the cytometer, granulocyte cells produce ___ FS and ___ SS; monocytes produce ___ FS and ___ SS; lymphocytes produce ___ FS and ___ SS.high; high ; high; low ; low; low
A graph used in FC is adot plot of FS versus SS

Section 3: Flow cytometry pt. 2

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The analysis software used in the project isBD FACSDiva (BD FACSCanto II is for obtaining flow cytometry data)

Section 4: Project details

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HTP flow cytometric analysis was carried out on ___ sperm from 4 donors (carried out to screen for the presence of 373 receptors).healthy/fertile
Among the 373 receptors, IL7-R (___) had low prevalence in healthy sperm samples. This receptor is known for its ___ function. (Its relatively low prevalence is compared with other markers of the 373 thing, but if you look at this same marker and compare fertile men with asthenozoospermic men, you'll find it even more decreased in the latter.)CD127 ; anti-apoptotic
In asthenozoospermic samples, there was a significant ___ in IL7R+ve spermatozoa.decrease
Aim #1 was to identify new extracellular markers for the isolation of rare ___ spermatozoa in ___ ___, thus avoiding ___. (Aim #2 was to do the same in NOA ___ ___.)functional ; NOA ejaculate ; mTESE ; (testicular biopsy)
CD52 (expressed on lymphocytes and epididymal epithelial cells) is AKAmaturation-associated sperm antigen
In one study, it was found that CD52 is ___ ___ for fertilization in the mouse, either _________ or _________.not required ; in-vivo ; in-vitro

Section 5: Andrology update: TEX101

Question Answer
An imaging flow cytometerAmnis ImageStream (optimal for 8 colors...max is 11)
Objective #1 of project is to confirm that testicular sperm have 2 ___ ___ and identify the ___ of the sperm contained within the ___.haploid peaks ; morphology ; peaks
Objective #2 is tot
CD143+ is expressed more in ___ sperm than in ___ spermtesticular ; ejaculate
Examples of well characterized exosomal markers?CD81, CD9, CD63
CD137 is of the ___ ___ protein family, and is involved in-TNF receptor ; immune cytolytic activity (enhances it)
CD137 is much more highly expressed in ___ samples compared with ___ ones.oligoasthenospermic ; normal
CD177 is similar in structure to theurokinase plasminogen activator receptor
During capacitation, ___ is released from spermvitronectin (VTN or VN)
CD177 is found in the ___ ___ and is involved in ___ ___ by binding to ___.extracellular matrix ; cell-cell interaction ; VTN
CD 177 is more highly expressed in ___ samples compared with ___ ones.normal ; oligoasthenospermic

Section 6: Updated info - retrieval strategies

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Microfluidic chips
lisin your obama

Section 7: Updated info - quality assessment

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Section 8: Updated info - patient subgrouping

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Section 9: Updated info - CD127 (IL-7R)

Question Answer
IL7R (or IL7 itself) regulatesB and T cell development, homeostasis and proliferation
IL7R has an ___ function.anti-apoptotic
IL7R is a ___ receptor, made up of a ___ chain and a ___ chain; the latter is a _____ for many IL receptors.heterodimeric ; IL7R-a ; IL7R-y ; common y chain
IL7 isthe most prominent cytokine in semen of healthy fertile individual
In rats, IL7 suppresses ___ cell ___, and promotes ___ maturation.granulosa ; apoptosis ; oocyte
IL7R is found on rat ___, ___ cells and ___ cells.oocytes ; granulosa ; cumulus
IL7R was detected on the surface of ___ to ___% of spermatozoa in normozoospermic samples.8 to 15 (but is significantly decreased in asthenozoospermic samples)
The finding that the IL7R is significantly ___ on the surface of spermatozoa from asthenospermic samples may be important in the mechanism underlying some cases when there are high levels of ___.lower ; necrospermia
When comparing the properties of IL7R+ve and IL7R-ve sperm fractions and DFI: L7R+ve was ___ ± 0.7 while IL7R-ve was ___ ± 3.4 (P = 0.012).1.5 ; 5.6

Section 10: Negative biomarker‑based male fertility evaluation: sperm phenotypes associated with molecular‑level anomalies

Question Answer

Section 11: IL7R project proper

Question Answer
m-IgGκ BPmouse IgG kappa binding protein (antibody we ordered)
The m-IgGκ BP is ___ to ___ ___.conjugated ; Horseradish Peroxidase (HRP)
HRP is often used in ___.conjugates (molecules that have been joined genetically or chemically)
HRP is used to determine the presence of a ___ ___, e.g. when an ___ is conjugated to HRP- this may be used to detect a ___ ___ of a specific protein in a ___ ___.molecular target ; antibody ; small amount ; western blot
In an antibody-HRP complex, he antibody provides the ___ to ___ the protein of interest, and the HRP ___, in the presence of a ____, produces a ___ ___.specificity ; locate ; enzyme ; substrate ; detectable signal
The IL-7R Antibody (G-11), another product we ordered, is a mouse ___ IgG1 (___ ___ ___).monoclonal ; (kappa light chain)
The G-11 is recommended for detection of IL-7R of _______ by _______.mouse, rat and human origin ; WB, IP, IF and ELISA

Section 12: MACS

Question Answer
The MACS micro-beads are so small they do not ___ cells or ___ cell surface epitopesactivate ; saturate
MACS MicroBeads are 50-nm ___ particles that are ___ to highly specific ___ against a particular ___ on the cell surfacesuperparamagnetic ; conjugated ; antibodies ; antigen
The MACS columns are designed for ___ ___. They can also be used for the ___ of strongly ___ ___ cells.positive selection ; depletion ; magnetically labeled
When placed on a magnetic separator, ferromagnetic spheres (which ___ the MACS ___) ___ the magnetic field. They produce a high ___ within the ___.(compose ; columns) ; amplify ; gradient ; column
Magnetically labeled cells are held in ___ within the column and do not actually “___” the column matrix. This ___ minimizes ___ on the cells and allows for efficient ___ ___ by avoiding cell ___.suspension ; bind ; suspension ; stress ; sterile washing ; aggregation
In indirect magnetic labeling, first the cells are labeled with a ___ antibody directed against a cell surface marker.primary
In indirect magnetic labeling (step two), the cells are labeled with microbeads which bind either the (___) ___ itself or a ___ that is ___ to it, e.g. a biotin-___ antibody or a ___-___ antibody.(primary) antibody ; molecule ; attached ; biotin ; fluorochrome-conjugated

Section 12.5: MACS - separation strategies

Question Answer
Positive selectionstandard thing
Depletion refers toremoving an unwanted cell type from the population (by labeling it w the microbeads) - same as positive selection minus the last step of eluting labeled cells
Untouched isolationsame as depletion but the purpose here is to deplete (=label) ALL other cell types and collect a (single?) particular kind of cells in the flow-through fraction
Sequential sortingA combination of two subsequent separations, applied to isolate cell subsets that can be distinguished from other cell types through their expression of two different markers
Sequential sorting is also useful forCell types for which a specific marker has not been defined.
Sequential sorting can be composed of eitherdepletion followed by poz selection or two subsequent poz selections
Depletion followed by poz selection is useful whenthe undesired cells and target cells have one marker in common

Section 13: ICC

Question Answer