eamag/protocols-with-steps huggingface dataset:
TITLE: Mouse Olfactory Horizontal Basal Cell Culture
AUTHORS: Alp Ozgun, Priya Suman, Josée Coulombe, Julianna Tomlinson, Earl G. Brown, John M. Woulfe, Michael G. Schlossmacher

[DESCRIPTION]
This protocol describes isolation and culture of basal cells from the olfactory epithelium of mice. The process involves serial enzymatic treatment of dissected olfactory epithelia and subsequent plating in commercial airway medium with dual SMAD inhibitors. The resulting cultures express the horizontal basal cell markers CK5 and CK14.

[STEPS]
1. Dissect olfactory mucosa from nasal septum on ice .
Induce anesthesia via intraperitoneal injection of 0.15 mL euthanyl, followed by
decapitation. Peel off skin and excise the eyes and the jaw. Disinfect the skull with 70% ethanol and carefully bisect along the sagittal plane, maintaining a 1 mm distance from the interfrontal suture. Transfer the larger segment of the skull under a dissection microscope, dissect a 1 mm distal section from the nasal bone. Carefully remove the nasal bones and collect the olfactory epithelium surrounding the septal cartilage into Hank's Balanced Salt Solution.


2. Mince the tissue in 2.7 mL dissection medium, add 300 µL 10x Collagenase/Hyaluronidase and transfer to a 15 mL falcon.

3. Briefly vortex falcon and rotate at 37 °C for 1h.

4. Spin down at 80 x g for 30s and discard supernatant.

5. Triturate pellet in 5 mL TrypLE for 2 min and add 10 mL cold HBSS.

6. Spin down at 350 x g for 5 min and resuspend cells in 1 mL warmed dispase and 50 uL DNase 1. Triturate for 2 min.

7. Add 10 mL cold HBSS and filter through 40 um cell strainer.

8. Spin down at 350 x g for 5 min, discard supernatant, resuspend in HBC medium with 10 micromolar (µM) ROCK inhibitor Y27632.

9. Plate on PDL and laminin coated wells, one mouse septum on 1.9 cm2 (1 well in a 24-well plate)
10. 10. Refresh medium the next day without ROCK inhibitor.
11. 11.  Feed every 2 days. Passage with accutase and do not use any other cell dissociation products such as TryplE of trypsin. Cultures are homogenously CK5+ after 3 passages.
2.
TITLE: New chemiphotobleaching protocol for Raman spectroscopy
AUTHORS: Elena Yakubovskaya, Tatiana Zaliznyak, Joaquin Martinez Martinez, Gordon Taylor

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We present a new and simple chemiphotobleaching method to irreversibly suppress background fluorescence of biological specimens during sample preparation for Raman microspectroscopic analysis. Our method combines mild hydrogen peroxide oxidation with broad spectrum visible light irradiation of the entire specimen. We show that >99% of the entire sample’s background fluorescence is eliminated after simultaneous exposure to a low concentration of hydrogen peroxide (3%) and irradiation by a standard photodiode lamp for 0.5–2 hours.This treatment permits observing intracellular distributions of macromolecular pools, isotopic tracers, and even viral propagation within cells previously not amenable to Raman microspectroscopic examination.</div></div>

[STEPS]
?. Concentrate cells from fixed suspension onto GTTP 0.2 um membranes by filtering appropriate volume. (depend on cells density)
?. Fix cells suspension with 2% final concentration of formaldehyde for 15 min.
?. Put the filter in a small petri dish and fill in with 2 ml of 3 % Hydrogen Peroxide.
?. Put the petri dish with the filter under bright white (3695) direct light (60 Lumens per Watt) (regular desk LED light lamp) for 0.5 – 3 hours. The distance between the light source and petri dish should be 5-10 cm.
?. Filter-Transfer-Freeze bleached sample to the mirror-finished stainless steel microscope slide (Taylor et al., 2017; dx.doi.org/10.17504/protocols.io.g4qbyvw):
?. Prepare filter wedges of GTTP membranes and mirror-finished stainless steel microscope slide:
?. Place 5 ul sterile MilliQ water on a clean mirror-finished stainless steel microscope slide and then wedge sample side down.
?. Place mirror-finished stainless steel microscope slide and filter on -80°C chilled aluminium block. After ~20 sec, peel membrane away and cells remain frozen to slide. Return slide to RT and air dry.
?. Perform Raman spectroscopy measurements.
TITLE: iPSC Freezing
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Aspirate media
?. Gently wash cells with 1x PBS.
Use 2-3 mL per well in 6 well plate.
?. Add Accutase (Gibco A11105-01) directly to the cells and incubate at for 3-4 minutes.
Individual donor cell lines exhibit variable sensitivity to accutase-mediated dissociation. Thus, monitor cells closely to determine when single cell dissociation is achieved.
For a 6 well plate, add 0.75-1 mL per well. For a 10cm2 dish, add 3 mL.
?. Add DMEM/F12 directly to cells.
?. Tap dish to aid in dislocation of cells.
?. Collect cells in conical tube (15mL/50mL depending on volume).
?. Add - DMEM/F12 to dish to remove all cells from the dish and add to conical tube.
2 ml
5 ml
?. Centrifuge cells at 750 rpm for  at room temperature.
?. Carefully aspirate supernatant.
To avoid aspirating cell pellet, it is OK to leave a small amount of media (0.5-1mL).
?. Resuspend cell pellet with mTesR1 (No Rock Inhibitor).Use volume appropriate for freezing. Assume 1 mL  per cryovial total and add ½ total volume of mTesR1. Pipet cells 1-2 times only to preserve cell clumps.
Example: to freeze 10 tubes, you will need 10 mL total and will add 5 mL mTesR1 to cell pellet (and 5 mL of 2x Freezing Media below).
?. Add an equal volume of cold 2x Freezing Media (20% DMSO, FBS). Pipet cells 1 time only to preserve cell clumps.
?. Transfer cell suspension to pre-labeled cryovials (1 mL per cryovial).                 -Cell Type                 -Line Name                 -Passage #                 -Date                 -Your Name
?. Freeze vials at in foam racks for 48-72 hours.
?. Transfer vials to liquid nitrogen for long-term storage.
TITLE: Ikaria Lean Belly Juice Australia
AUTHORS: it makes weight reduction simpler by empowering thermogenesis What are the Benefits of Ikaria Lean B, it is smarter to comprehend the outcomes might be slow.  Where To Buy Ikaria Lean Belly Juice in Aus, eBay, or Walmart.Silymarin: It is known for helping digestion. Otherwise called milk thorn, it is a characteristic liver-supporting spice. It assists with killing uric corrosive and toxins fro, organ purging, and liver health. It safeguards synapses against oxidative harm and forestalls cognitive deteriorati

[DESCRIPTION]
Ikaria Lean Belly Juice Australia
http://www.bestsupplement24x7.com/supplemet/ikaria-lean-belly-juice-australia/
https://proton-4.jimdosite.com/
https://www.facebook.com/IkariaLeanBellyJuiceAustraliaAU/
 https://the-dots.com/projects/ikaria-lean-belly-juice-australia-774691
https://www.spreaker.com/show/ikaria-lean-belly-juice-australia_2
https://www.pinterest.com/ikarialean/httpwwwbestsupplement24x7com/
https://www.linkedin.com/pulse/ikaria-lean-belly-juice-australia-tomy-zonmy/
https://medium.com/@tomyzonmy/ikaria-lean-belly-juice-australia-8d63e98387a3
https://tomyzonmy.clubeo.com/news/2022/06/07/ikaria-lean-belly-juice-australia?_ga=2.97570719.1841894455.1654614461-365148188.1654614459

[STEPS]
1. Ikaria Lean Belly Juice Australia
http://www.bestsupplement24x7.com/supplemet/ikaria-lean-belly-juice-australia/
https://tomy-zonmy.jimdosite.com/
https://the-dots.com/projects/ikaria-lean-belly-juice-australia-777369
https://medium.com/@monticarlon/ikaria-lean-belly-juice-australia-3fe9d84abd25
https://tomyzonmy.clubeo.com/news/2022/06/11/ikaria-lean-belly-juice-australia?_ga=2.229941343.2104291137.1654938972-902855542.1654938971
https://www.facebook.com/IkariaLeanBellyJuiceAustraliaAU/
https://www.spreaker.com/show/ikaria-lean-belly-juice-australia_2
https://www.pinterest.com/ikarialean/httpwwwbestsupplement24x7com/
https://www.linkedin.com/pulse/ikaria-lean-belly-juice-australia-tomy-zonmy/
2. 
3.
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Transfection by Electroporation in Euplotes crassus
AUTHORS: Angela Piersanti

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. 2x104 Euplotes crassus cells were collected and resuspended in 0.3 M glucose solution (7% sea water and 93% of 0.3 M glucose solution).
?. Each round of transfection 250 µl of cells were used. 0.25 µg of Label IT® Plasmid Delivery Control Cy®3 (Mirus) were added alone or mixed with 2.5 µl of Lipofectamine® 2000 Transfection Reagent (Invitrogen) according to the supplier.
?. The sample was transferred to the 0.2 cm cuvette. Bio-Rad Gene Pulser was used. Conditions were set as follows: 0.2 kV, 25 µFD, 100 Ω. Time constant around 1.2.
?. More than 50% of cells were viable after electroporation (few cells fused together), then cells were resuspended in 3 ml of sea water.
?. The plasmid was visible in the cytoplasm immediately after the electroporation by fluorescent microscope.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>RNA extraction from E. coli cells based on the method described by Chomczynski and Sacchi, 1987</p>

[BEFORE_START]
<p>Always keep your samples on ice!</p>

[GUIDELINES]
<p>RNA is sensitive to degradation! Wear gloves, keep samples on ice when possible, use filter-tips<br />and RNase free reagents. Pre-cool centrifuges and store isolated RNA-samples immediately at -20<br />or -80°C.</p>

[STEPS]
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TITLE: Single cell analysis of iPSC-derived midbrain organoids
AUTHORS: María José Pérez J., michela.deleidi

[DESCRIPTION]
The following script was used for analysis of gene corrected (GC) versus GBA1 mutant (MUT) midbrain organoids. The purpose was to combine, filter, integrate, and identify clusters and differentially expressed genes sets. 

[GUIDELINES]
This script is based on the Seurat tutorials
https://satijalab.org/seurat/articles/integration_introduction.html
https://ucdavis-bioinformatics-training.github.io/2019-single-cell-RNA-sequencing-Workshop-UCD_UCSF/scrnaseq_analysis/scRNA_Workshop-PART1.html

[STEPS]
SECTION: Part 1. Data preparation
1. 
 

SECTION: Part 2. Quality control
2. 
 

SECTION: Part 3. Data preparation and normalization
3. 
 

SECTION: Part 4. Data integration and visualization (directly after Part 3)
4. 
 

SECTION: Part 5. Obtain information from the datasets
5. 
 

SECTION: Part 6. Merge and analyse subclusters
6. 
 

SECTION: Part 7. Create a subset of cells from a selected cluster to reanalyze
7. 
 

SECTION: Part 8. Differential expressed genes using FIndMarkers with MAST
8. 
 

SECTION: Part 9. Analysis of differential expressed genes using FIndMarkers with MAST in non-integrated samples (directly after Part 3)samples (directly after Part 3)
9.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The current protocol is a bleeding-resuscitation model intended to imitate the effects of severe blood loss and subsequent fluid resuscitation. The experimental subjects were Vietnamese pot-bellied pigs of both sexes weighing 33±4 kg. The animals underwent a 12-hours fasting pre-operatively but had free access to water. Anesthesia was induced by intramuscular injection of a mixture of ketamine (20 mg/kg) and xylazine (2 mg/kg) and maintained with a continuous iv. infusion of Propofol (50 µL/min/kg IV; 6 mg/kg/hr), while analgesia was maintained with intermittent nalbuphine (0.1 mg/kg). After endotracheal intubation, the animals were mechanically ventilated with a Hamilton C1 respirator (Hamilton Medical AG, USA). The tidal volume was set to 10 ml/kg, and the respiratory rate was adjusted to maintain the end-tidal carbon dioxide and partial pressure of arterial carbon dioxide within the range of 35–45 mmHg and the arterial pH between 7.35 and 7.45. The depth of anaesthesia was assessed by checking jaw tone. After induction of anaesthesia, catheters were inserted into the left jugular vein, left external carotid artery and the left femoral artery. For invasive hemodynamic monitoring, a transpulmonary thermodilution catheter (PiCCO, PULSION Medical Systems SE, Munich, Germany) was used. The femoral artery served as the site for arterial blood gas sampling, the central venous line was used for taking central venous blood gas samples and for the injection of cold saline boluses for the thermodilution measurements, whilst the carotid arterial catheter was used for draining blood. A transpubic catheter was placed into the urinary bladder for monitoring renal function. Animals were covered in scrubs and an external heating device was used to maintain physiological body temperature.</p>

[STEPS]
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TITLE: Genetic network and data analysis
AUTHORS: Joachim Nwezeobi, Onyeyirichi Onyegbule, Chukwuemeka Nkere, Joseph Onyeka, Sharon van Brunschot, Susan Seal, John Colvin

[STEPS]
?. The alignment file (.nex) obtained from Geneious was used to build the minimum spanning tree using Phyloviz [51], to identify the relationships and mutational distances between the different haplotypes.
?. After the random selection and testing of two whitefly samples per field, a two-sided Fisher test and the chi-square test were carried out on the number of different populations identified in the two samples to ascertain whether or not more sampling was needed to capture the entire diversity of the genetic groups present in the survey collections.
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Luminex Milliplex Cytokine/Chemokine 9-plex MAG manufacturer's protocol</p>

[STEPS]
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TITLE: P1 20/05
AUTHORS: Maria  Gul

[DESCRIPTION]
qa

[STEPS]
1. test 

Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Nam libero tempore, cum soluta nobis est eligendi optio cumque nihil impedit quo minus id quod maxime placeat facere possimus, omnis voluptas assumenda est, omnis dolor repellendus. Morbi imperdiet, mauris ac auctor dictum, nisl ligula egestas nulla, et sollicitudin sem purus in lacus. Nam quis nulla. Nunc auctor. Fusce wisi. Aliquam erat volutpat. Curabitur ligula sapien, pulvinar a vestibulum quis, facilisis vel sapien. Cras pede libero, dapibus nec, pretium sit amet, tempor quis. Nullam dapibus fermentum ipsum.
2. test 2

Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Nam libero tempore, cum soluta nobis est eligendi optio cumque nihil impedit quo minus id quod maxime placeat facere possimus, omnis voluptas assumenda est, omnis dolor repellendus. Morbi imperdiet, mauris ac auctor dictum, nisl ligula egestas nulla, et sollicitudin sem purus in lacus. Nam quis nulla. Nunc auctor. Fusce wisi. Aliquam erat volutpat. Curabitur ligula sapien, pulvinar a vestibulum quis, facilisis vel sapien. Cras pede libero, dapibus nec, pretium sit amet, tempor quis. Nullam dapibus fermentum ipsum.
TITLE: Retro-orbital Bleeding for Rats
AUTHORS: Sharona Sedighim, Olivier George

[STEPS]
?. [Eye Bleeds Procedure]
Take a microcapillary tube (broken in half) and place it behind the eye.
?. [Eye Bleeds Procedure]
Twist it until you hear/feel a little pop, and blood should start coming out.
?. [Eye Bleeds Procedure]
Let blood drip into the purple collection tube (try to fill it to the top line).
?. [Eye Bleeds Procedure]
Once you complete the collection, remove the microcapillary tube and clean the eye with a cotton tip applicator and/or gauze.
?. [Eye Bleeds Procedure]
Put the cap on the tube, and slowly flip tube up and down so the blood goes on all sides of the tube (prevents it from clotting).
?. [Eye Bleeds Procedure]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Concentrate phytoplankton samples about 100-fold typically from 5L down to 20 mL.&nbsp; Takes about 1 hour&nbsp;per sample. &nbsp;Samples can be used for flow cytometry sorting or for cultures.&nbsp; Enrichment by TFF usually keep growing for a longer time than unconcentrate samples.<br /><br /><strong>Reference<br /></strong><a href=".https://www.researchgate.net/publication/42254387_Use_of_flow_cytometric_sorting_to_better_assess_the_diversity_of_small_photosynthetic_eukaryotes_in_the_English_Channel" target="_blank">Marie, D., Shi, X.L., Rigaut-Jalabert, F. &amp; Vaulot, D. (2010). Use of flow cytometric sorting to better assess the diversity of small photosynthetic eukaryotes in the English Channel. <em>FEMS Microbiology Ecology</em>. 72. p.pp. 165&ndash;178<strong>.<br /></strong></a>

[BEFORE_START]
<a href="https://s3.amazonaws.com/pr-journal/qqchcw.jpg" target="_blank"><img src="https://s3.amazonaws.com/pr-journal/qqchcw.jpg" alt="" /></a><br /><strong>Necesssary equipement</strong><br />&bull;&nbsp;&nbsp;&nbsp; Vivaflow Cartridge 100,000 MWCO (Regenerated Cellulose - RC) VF20C4&nbsp; for viruses<br />&bull;&nbsp;&nbsp; &nbsp;Vivaflow Cartridge&nbsp; 0.2 &mu;m (PES) VF20P7 for plankton<br />&bull;&nbsp;&nbsp; &nbsp;Masterflex Pump 6-600 rpm (ref Bioblock F39671)<br />&bull;&nbsp;&nbsp; &nbsp;Rotor 3 &ldquo;galets&rdquo; (ref&nbsp; F39110)<br />&bull;&nbsp;&nbsp; &nbsp;1 Head high throughput (ref F40103) (Can be replaced with quick load head)<br />&bull;&nbsp;&nbsp; &nbsp;Replace tube provided by stronger tube with two connectors (see picture)<br />&bull;&nbsp;&nbsp; &nbsp;Bottle 6 L<br />&bull;&nbsp;&nbsp; &nbsp;Bottle 1 L<br />&bull;&nbsp;&nbsp; &nbsp;Conical tube 50 mL<br />&bull;&nbsp;&nbsp; &nbsp;Masterflex Tygon tubing size 16<br />&bull;&nbsp;&nbsp; &nbsp;Plastique pipettes (1 mL)<br />&bull;&nbsp;&nbsp; &nbsp;Clamps with screw (to control retentate speed)<br />&bull;&nbsp;&nbsp; &nbsp;Clamps

[GUIDELINES]
In order to estimate actual concentration efficiency measure Synechoccus, pico, and nanoeularyote concentration before and after TFF.

[STEPS]
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TITLE: Size Exclusion Chromatography with Multiangle Light Scattering (SEC-MALS)
AUTHORS: Minghao Chen, Xuefeng Ren

[DESCRIPTION]
SEC-MALS

[STEPS]
1. Concentrate the purified protein sample to 4-5 mg/ml

2. Instruments:
Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA)
Wyatt DAWN HELEOS-II MALS instrument
Wyatt Optilab rEX differential refractometer (Wyatt, Santa Barbara, CA)
WTC-050S5 size-exclusion column (Wyatt) 
40 ul sample loop


3. Equilibrate the system and column with buffer containing:
25 mM HEPES pH 7.5, 200 mM NaCl, 1 mM MgCl2, 2 mM TCEP
  


4. Turn on the laser 1 hour before the experiment.
Confirm the readout is stable 
5. Run 5 mg/ml BSA to calibrate the system
6. Inject the sample and run (takes ~1 hour/sample)
7. Analyze the results by ASTRA V software (Wyatt). 
MALS signals, combined with the protein concentration determined by refractive index, are used to calculate the molecular mass of the complex.
TITLE: Almond Macaroons Recipe
AUTHORS: Lenny Teytelman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is one of the EASIEST deserts to make.  It takes almost no time, bakes quickly, and has no butter, wheat flour, or egg yolk.  It is also infinitely flexible - add Khalua instead of vanilla, make them chocolaty with a quarter cup of cacao, place an almond inside each one, or substitute almond with shredded coconut (partially or completely).</div><div class = "text-block">This is inspired by Mark Bittman's macaroons recipe. </div><div class = "text-block"><span>Bittman, Mark. (2008). </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">How to cook everything. 2,000 simple recipes for great food</span></a><span>. Hoboken, NJ :J. Wiley</span></div></div>

[STEPS]
?. Preheat oven to  degrees.
350 °F
?. Mix almonds, almond meal, and sugar in a bowl.
?. Add egg whites and vanilla, and mix thoroughly to make batter.
?. Bake for , until slightly browned.
?. Cool on racks.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>This is a short tutorial on the basics of getting started with standalone BLAST+ in the Ubuntu command line.</strong></p>
<p>Code is intended for use on an Ubuntu 16.04 LTS OS.</p>
<p> </p>
<p>Note that a web server for BLASTP is also available: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&amp;PAGE_TYPE=BlastSearch&amp;LINK_LOC=blasthome</p>
<p> </p>
<p>For details on BLAST please see the NCBI webpage and release notes: https://www.ncbi.nlm.nih.gov/books/NBK131777/</p>
<p>And the NCBI paper on BLAST+: https://www.ncbi.nlm.nih.gov/pubmed/20003500?dopt=Citation</p>

[STEPS]
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TITLE: https://rogerdselders.clubeo.com/news/2022/07/08/watch-this-exipure-reviews-instant-energy-like-the-latest-alive
AUTHORS: cyrusdwelch  

[DESCRIPTION]
Therefore, you won't need to fear approximately gaining weight and affected by the result of obesity. Before transferring in addition, knowing more about the substances present in Exipure Reviews is a need to.

[STEPS]
TITLE: Deep hind paw incision model
AUTHORS: Fanglin Lu

[DESCRIPTION]
The deep hind paw incision model is established to evaluate postoperative pain.
Based on TJ Brennan's rat model of incisional pain (1996), several modifications are made.
This protocol outlines the procedures from a to data anesthesia to consciousness recovery. 

[GUIDELINES]
Please follow the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

[STEPS]
SECTION: Anesthesia and disinfection
1. The mouse placed in a prone position under sevoflurane anesthesia (4 volume% in 1 L/min oxygen) delivered via a nose cone. Sevoflurane inhalation is used, as it is safe, non-invasive, rapid, and easy to control. Adequate anesthetic depth is ascertained by loss of response to tail clamp. 
SECTION: Deep hind paw incision
3. Make a 5-mm longitudinal incision through the skin using a scalpel blade, starting 2 mm from the proximal edge of the heel and extending toward the toe. 
  

SECTION: Deep hind paw incision
4. Elevate the underlying plantaris muscle using curved iris forceps, with the help of a double-ended micro spatula.
Incise the fascia and plantaris muscle with angled 90° dumont forceps.
Keep the position for 5 min. 
  

SECTION: Deep hind paw incision
5. After hemostasis, close the wound using a 6-0 nylon suture and cover it with bacitracin ointment to prevent wound infection. 
  

SECTION: Deep hind paw incision
6. Place the mouse in a recovery cage. Once the mouse is fully recovered as it regains the righting reflex and ability to stand, return it to its home cage. Respiratory patterns and reactions to surgical stimulation are continuously monitored during the entire surgical procedure. 
 

SECTION: Anesthesia and disinfection
2. Keep the ipsilateral paw in plantar-flexion position. Disinfection with 10% povidone–iodine solution. Place a drape.
 

SECTION: Sham surgery
7. The sham surgery comprises anesthesia, antiseptic preparation, and application of topical antibiotics without skin and muscle incision and elevation.
TITLE: Determination of C3 concentration by the Mancini test.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. An appropriate anti-C3 antiserum (antibody) is poured in the center well of an agar-containing plate.
?. Carefully circular wells are cut and detached from the plates.
?. A series of standards containing known concentrations of C3 are placed in separate wells, while “unknown” human serum samples and control are placed in other wells.
?. A ring of precipitate forms in the area of optimal antigen-antibody concentration, meaning anti-C3 - C3 reaction as the antigen diffuses radially.
?. The diameters of the rings are measured and perceived normally in 48-72 hours.
?. Finally, a standard curve is developed using the ring diameters of the standards versus the concentrations.
?. A curve is then used to plot the concentration of the control and unknown C3 samples.