Research: Immunotherapy Flashcards
Project Immunotherapy - Isolating PBMC - Isolating macrophages - Differentiation and maturation - Purity test with FACS - ELISA
Blood consists of 55% plasma and 45% blood cells, the most prevalent cell in the cullular population is (1) and can be separated from plasma with (2). The buffy coat is then found at (3).
(1): erythrocytes
(2): centrifugation
(3): thin layer between erythrocytes and plasma
When counting cells in a counting chamber, how is the dilution made when the final dilution is 20x?
10x dilution first, then 1:2 with tryphan blue
> use as little trypan blue as possible as it is expensive
Trypan blue will colour the …. cells in counting
Dead cells
> in cytosol, trypan blue colors proteins
> live cells have an intact impermeable plasma membrane: trypan blue cannot enter the cell
> dead cell: permeable plasma membrane
When using the counting chamber:
> Pipetting
> Counting
> Calculate
- Pipette under glass
- Count 25 squares: 2 rows + 1 square = 0.1 ul
> cells / ml > count in 25 squares * 10000 (0.1 ul to 1 ml) * dilution factor > (in 10^6 cells / ml) - Viability cut-off 70%
- count inside square and two inner borders
Cleaning counting chamber
- Before: alcohol
- After: first demiwater and then alcohol, otherwise cells are stuck (keep glass on)
Why resuspend before pipetting cell suspension into counting chamber
They are heavy and sink to the bottom.
In the practical, dendritic cells were matured with 5 maturation mixes, why?
To test their potential to gain mDCs which promote Th1 response and migration efficiency to LNs
> different maturation mixes lead to different mature DCs
Explain Immunotherapy based on DCs
Patient
> Derive and isolate mononuclear cells from blood
> Isolate monocytes (there are very few DCs in blood for immunotherapy)
> differentiate to iDCs (with GM-CSF and IL-4)
> Mature with maturation mixes
> antigen loading: load antigens to MHC-I/II
> injection into patient
DC immunotherapy: functions DCs
Antigen presentation > T-cell activation
Characteristics of DCs for optimal use in Immunotherapy
Characteristics of mDC for functional in immunotherapy
> Correct antigen presentation on MHC-II: activate (CD4+) T-cells
> Express co-stimulatory molecules: CD40
> produce proper cytokines for correct T-cell polarization
> migration capacity of DCs is needed to travel to lymph nodes of T-cells
» in practical: last two tested
MoDCs of patient are used, why
Monocyte Derived dendritic cells (MoDCs)
> from patient: personalize epitope targeting, make therapy more powerful
> reduce risk of rejection
Why Th1 response in immunotherapy? Name the functions.
- Induce CTL response for tumor killing
- Eradication intracellular pathogens by macrophages
Which cytokine is needed for Th1 polarization, and was thus measured in the ELISA
IL-12
Why is IL-6 measured in ELISA?
To detect the DC activation
> IL-6 as control for any cytokine production
> if no IL-6 produced, then there will be no IL-12 produced.
> IL-6 production without IL-12 production is possible, but then the unwanted CD4+ T-cell polarization is induced by the DCs
Migration capacity of DCs of immunotherapy important, why
They need to migrate to lymph nodes to activate CD4+ T-cells with MHC-II
> certain stimuli can increase migration capacity of DCs
> by stimulating receptor expression in DCs
> Chemokine receptors
» antigen presentation and T-cell activation takes place in LN (whereas in periphery: TLR triggering and antigen uptake)
What if the matured DCs make IL-12 but have no migration capacity
Not functional, cannot reach LNs to activate T-cells
Name the cell types in buffy coat and if they stay above or under Ficoll (higher density) after centrifugation
Lower density: stay above: T-lymphocytes, B-lymphocytes, NK cells, Monocytes, Thrombocytes/platelets (in PBMC ring fraction)
Higher density: stay under: granulocytes, erythrocytes
> granulocytes lay on top of erythrocytes
Normal fractions in PBMC fraction
Thrombocytes are removed during multiple wash steps
> T-lymphocytes 64%
> B-lymphocytes 8%
> NK cells 8%
> Monocytes 20%
6 steps of Sandwich ELISA (we have done this)
- Coat the wells of a plate with a specific antibody directed against the specific cytokine (or antigen of interest)
- Incubate the culture supernatant in the plate
- Incubate with biotin-labeled detection antibody specifically directed against the cytokine
- Add streptoavidin-HRP (Horseradish peroxidase), binding to biotinylated antibodies
- Add colorless substrate (Tetramethylbenzidine - TMB) which in combination with H2O2 is converted by HRP into a blue compound
- Stop the reaction using H2SO4
Difference between the primary and second primary antibody in Sandwich ELISA
- Different binding epitope on the antigen of interest
- Second primary antibody is bound by biotin at Fc region > Streptavidin-HRP can bind the biotin molecule
In ELISA the color strength represents the …
concentration of antigen of interest
Advantages and disadvantages Sandwich ELISA
- High sensitivity
- High specificity
However
» Sensitivity: the ability of a test to correctly identify patients with a disease/ true positives. Specificity: the ability of a test to correctly identify people without the disease/ true negatives - Cross-reactivity between the antibodies, which could cause a high background signal (because not washed away!)
- More expensive
- Longer procedure
Direct ELISA
- Antigen of interest is coated to the plate
- Primary antibody with biotin at Fc binds
- Streptavidin-HRP
- TMB and H2O2
- Stop with H2SO4
When is Direct ELISA used?
When only one antibody is available for the antigen of interest
Advantages and disadvantages of Direct ELISA
- Fast and simple
- Less prone to errors
- Less cross-reactivity
However - Less specificity
- Minimal signal amplification (during coating, other antigens not of interest bind to the well and less antigens of interest are able to also bind to the plate)
ELISA when interest in an antibody
Direct ELISA
> plate directly coated with the antibody as well as antigens not of interest
primary antibodies vs secondary antibodies: when used?
Primary: bind the antigen directly (in Sandwich and direct ELISA, can be bound to biotin)
> however, specific antibodies bound to biotin are not always available
> secondary antibody used
Indirect (sandwich) ELISA
When: no biotinylated antibody targeting the antigen of interest is available
( Coating with primary antibody for antigen if sandwich)
- Antigen of interest added
- Primary antibody (with different epitope specificity than coating)
- Secondary antibody specific for primary antibody added which is biotinylated
- add Streptavidin-HRP
- add TMB and H2O2
- stop with H2SO4
Secondary biotinylated antibodies for ELISA bind to:
Fc tail of primary antibodies used
> do not bind one specific antibody, but antibodies of one specific species
Advantages and disadvantages of Indirect sandwich ELISA
- Sensitive
- Cheaper
However - Less specificity
- Cross-reactivity between primary and secondary antibody
- Longer procedure
Competitive ELISA
When: Impure samples
- Primary antibody is first incubated with the antigen
- Then, antigen-antibody mix is added to well which is coated with same antigen of interest
> if concentration of antigen was high in pre-incubated sample, they cannot all bind (less freely antibodies left)
> signal will be less strong
- Wash
- Secondary biotinylated antibody
- Streptavidin-HRP
- TMB H2O2
- Stop H2SO4
Advantage and distadvantage competitive ELISA
- Use impure samples
- Robust and consistent
However - Less specificity
3 differences between primary and secondary antibodies in ELISA
- Primary binds the antigen specifically, the secondary binds the primary its Fc
- Derived from different species
- Different role in detection: primary must recognize antigen for specificity and secondary must amplify signal because conjugated to biotin label which is recognized by streptavidin-HRP
- Concentration of primary antibody used is lower because it is more specific and the secondary is for amplification.
1 A virologist wants to detect a viral antigen in a tissue sample. There is no biotinylated antibody available.
2 A farmer uses a pesticide to treat crops. Regulatory agencies need to ensure that the pesticide residues on the harvested crops are below permissible levels to ensure the safety of consumers. However, the harvested crop sample contains a mixture of various organic and inorganic substances.
3 You want to measure the level of the cytokine IL-12 in culture medium produced by dendritic cells. You have two different antibodies directed against IL-12. One of them is biotinylated.
» which ELISA to use
1 > indirect ELISA
2 > Competitive ELISA
3 > Sandwich ELISA
How to calculate the concentration of the different dilutions of the concentration curve and the samples? 1. How to make the formula?
Concentrations of standard curve is known
> Raw values of standard curve taken and average of the duplo taken for known concentration
> insert blank value as well and take average
> make a graph of the OD (extinction value) against the concentration (in pg/ml or mg/ml etc) (with dots)
> create curve
> upper plateau not included (reaction is saturated) and lower plateau (too little conversion for signal) not included (only steep part taken)
> all values of standard curve should be at least 2 x blank value
> new plot with steep part made and trend line added
> enable formula
> change the formula so that concentration is put out in extinction
> Y = aX + b > X = (Y-b) / a
How to calculate the concentration of the different dilutions of the concentration curve and the samples? Calculate concentrations
- Calculate average extinction values of sample dilutions
- add averages in formula
- multiply by dilution factor
How to calculate the concentration of the different dilutions of the concentration curve and the samples? Inclusion and exclusion; choose right dilution
Exclude
- check extinction values of sample
> at least 2 x blank
> within standard curve steep line (extrapolating is not allowed, you do not know how the curve behaves outside the data points)
Include
> when multiple left for one sample, choose the one with the extinction closer to the centre of the calibration curve, this one is the most reliable
» when all concentrations have higher extinctions than the standard curve, then we know the concentration is higher than the concentration found in the highest point of the steep part of the standard curve, multiplied by the highest dilution factor
» when all concentrations have lower extinctions than the standard curve, then we know the concentration is lower than the concentration found in the lowest point in the steep part of standard curve, multiplied by the lowest dilution.
» for example, all above and highest dilution is 125 and highest concentration of steep standard curve is 5 mg/ml than it is at least 125*5 = >625 mg/ml
How to make Monocyte derived Dendritic Cells (MoDCs) in lab
Add granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 to monocyte isolate
How are monocytes isolated in a PBMC fraction with magnetic cell separator (MACS)?
Use CD14 microbeads (positive selection)
> Column is placed in MACS
> Washed with MACS buffer> flowthrough in waste
> wait for flow to end
> add cell suspension after resuspension: cells attached to an antibody-labeled magnetic bead will be pulled to the magnet and will not flow through: flowthrough in waste
> Rinse with MACS buffer multiple times
> column removed and placed on sterile tube > culture medium added and plunger used to harvest flowthrough
How is change in morphology (during DC maturation) observed?
Using a microscope
> imaging
> drawing
> write down confluence (percentage of cell coverage on the growth surface, ensure healthy cell growth) and viability (to identify stress, contamination or overgrowth)
Laminar air flow cabinet use
UV light on > kill all cells
> turn of UV light
> turn on light
> screen to cleaning mode: clean with ethanol
> set to work mode
> put materials in flow cabinet but disinfect them first with ethanol
> place next to each to not disrupt flow from back to front
> space in middle for working
> rest elbows when working
> nothing into waste bottle, pipette on distance
> blood working: gloves only in flow cabinet.
Migration in vitro measurement with transwell migration assay
- Permeable membrane separates the tranwell from the well
- cells are seeded in transwell and chemoattractant in well
- incubation
- migration possible, only cells which are responsive to the chemoattractant
- some cells can diffuse without additive
> negative control should be added to establish minimal migration capacity of the cells without additive
> 100% migration should be established - Quantification: remove transwell and analyze the well > count cells with microscope or by staining
100% migration and minimal migration capacity controls
- 100%: add cells in well directly, without transwell
- minimal migration capacity: add no additive / chemoattractant
Which chemokine is used to check the migration capacity of neutrophils?
CCL-21
Will there be cells migrating in the negative control of transwell migration assay
Yes, due to diffusion, this is the minimal migration capacity of the cells
Buffy coat used in the experiment was …
Diluted in PBS / 0.4% TNC
Ficoll-Hypaque function
Has known density (1.077 g/ml) that separates the buffy coat
Isolated PBMC ring fraction is washed, how?
Fill tubes with PBS/0.4% (with PBMCs)
> centrifuge
> decant supernatant
In the protein buffer for PBMCs, PBS + 4% HSA + 0.4% TNC was used, why?
HSA: Human Serum Albumin: provide necessary nutrients and promote cell growth
After PBMC isolation, the … is needed
Counting
> determine concentration and viability
> set amount of cells needed in cell suspension for monocyte isolation
Why is it important to wait until the buffer has almost completely run through the column before adding more buffer?
To ensure complete washing and removal of non-specifically bound cells and debris.
To maximize the purity of the target cell population.
»
Waiting ensures that the entire column has been washed, helping to get rid of cells or particles that are weakly bound.
By allowing the buffer to completely run through, it ensures the removal of non-target cells, increasing the purity of the isolated cells.
Why incubatio monocytes with CD14 (monocyte marker) microbeads at 4 degrees?
The recommended incubation temperature is 2–8 °C. Higher temperatures and/or longer incubation times may lead to non- specific cell labeling
Why is EDTA in MACS buffer
EDTA (ethylenediaminetetraacetic acid) is a chelating agent that binds divalent metal ions such as calcium and magnesium
> to bind divalent cations: the magnetic beads
> By chelating these cations, EDTA prevents cells from clumping together (aggregation) and helps to maintain their single-cell suspension, ensuring smooth separation during the MACS procedure. This is especially important when isolating specific cell types or when using magnetic beads to sort cells, as aggregation can interfere with the accuracy of cell separation
The Cellgro medium for the monocyte isolate consists of Cellgro medium + penicillin + streptomycin, why are these antibiotics added
To prevent bacterial growth
> prevent contamination and maintain aseptic conditions
Why do the monocytes elute from the column when placed in the elution tube?
The column is not placed in the magnet anymore,
Which samples were included in FACS purity test?
- Unstained PBMCs > baseline for gating
- PBMCs: stained
- Monocytes: stained
Why are samples for FACS incubated in the dark?
to prevent the fluorescence signals from photobleaching. Many flow cytometry experiments use fluorescently labeled antibodies or other markers to stain cells. These fluorophores are sensitive to light, and prolonged exposure to light, especially intense or UV light, can cause them to lose their ability to fluoresce, a phenomenon known as photobleaching.
Role of PFA in FACS procedure
Fixation of cells
> PFA: Paraformaldehyde
> cross-linking structure
Expectation FACS purity test
PBMC unstained
PBMC
Monocyte isolate
Monocytes NK-cells T-cells B-cells
Sample 1 0% 0% 0% 0%
Sample 2 20% 8% 64% 8%
Sample 3 90 1% 8% 1%
IL-4 and GM-CSF function
Induce differentiation monocytes to (immature) DCs.
Why is PBS added to the surrounding wells of the differentiating monocytes?
To reduce evaporation of culture medium
Why incubation monocytes for differentiation with 37 degrees and CO2
Replicate in vivo environment
For maturation mixes, the following ingredients were used in different combinations, name their function
> FCS
> MPLA
> LPS
> IFN-y
> TNFa
> IL-1b
> PGE2
> FCS: fetal calf sera: containing antigens
MPLA: monophosphoryl lipid A, detoxified LPS, potent adjuvant activity while no toxicity
LPS: bacterial cell wall component, trigger TLRs
IFN-y: induces maturation, also Th1 specific cytokine
TNFa: favors maturation
IL-1b: induces dendritic cells to produce IL-12
PGE2: Prostaglandin E2: enhances CCR7 expression in DCs (increase migration capacity)
Which chemokine receptor needs to be upregulated in the MoDCs to be attracted to CCL-21 in the transwell migration assay?
CCR-7
Why are MoDCs prepared in IMDM medium with FCS (fetal calf serum) for the migration assay?
To activate them with antigens
Why are stickers placed on ELISA plates?
To prevent it from drying out
H2SO4 stop solution
Used in ELISA utilizing TMB substrate solution with horseradish peroxidase (HRP) which colors it blue.
> sulfaric acid acts to inactivate the peroxidase enzyme by pH lowering.
> same amount of TMB and H2SO4 are added
> colors yellow: oxidized TMB colors yellow
Tween 20 is used in ELISA wash buffer, why
Emulgators
> removal of membranes or proteins
Een collega heeft op het lab muis-anti-CD3 toegevoegd maar heeft in de tweede stap
in plaats van Schaap-anti-muis beads Schaap-anti-konijn beads toegevoegd (bij MACS, magnetic activated cell sorting) Hoe los je dit op?
De secundaire antilichamen met microbeads zijn zwaar en komen na centrifugeren in het pellet terecht, en zijn zo dus niet te scheiden.
Het is verstandiger om alle ongebonden beads eerst door de MACS magneet te laten lopen na het wassen van deze met MACS buffer.
Eluteer daarna de losse beads.
> voeg daarna de juiste beads toe aan de cellen zonder de beads en ga verder met de procedure.
Na incubatie met beads bekijk je de celsuspensie onder de microscoop.
0.3p d. Waarom is het belangrijk dat je ook losse ongebonden beads ziet? (bij selectie T-cellen met beads)
Zodat je weet dat alle cellen van interesse gebonden zijn door een microbead, aangezien er ook ongebonden rondzweven.
> concentratie cellen van interesse in isolaat wordt gemaximaliseerd
Tijdens het kweken werk je steriel. Welke infecties kun je in je kweek
krijgen? (Noem er drie)
- Bacteria
> gram positive: Staphylococcus aureus, Streptococcus pneumoniae
> gram negative
> Mycoplasmas - fungi
> candida albicans
> aspergillus fumigatus - viral
- endotoxins
Beschrijf hoe je T cellen kunt isoleren uit de PBMC fractie met behulp van
negatieve selectie. Noem in je antwoord ook de CD markers waar je gebruik
van maakt.
In de PBMC fractie zitten T-cellen, B-cellen, monocyten en NK cellen
Negatieve selectie, selecteer voor de cellen die je wilt excluderen
> B-cellen: CD19
> NK-cellen: CD16
> Monocyten: CD14
Dan houd je CD3+ T-cellen over
Why brake at lowest setting when Ficoll density gradient separation
Be careful, do not shake your sample, you do not want the layers to be disturbed. This is also the reason why we use the lowest brake setting
MACS separation of blood cells: positive selection for monocyte
Add anti-CD14
> centrifuge and wash away unbound antibodies
> add secondary antibody conjugated to magnetic bead, like goat anti-mouse antibody
Disadvantage negative selection
If one of the bead-target cells did not bind the bead, it is not selected and ends up in the cells of interest part, while the labeled cells are isolated with high purity.
Why, in negative selection, does just one type of secondary antibody have to be added
All bind Fc domain of mouse anti-CD? for example
