Standard protocols, ready for the bench
52 step-by-step protocols across immunology, molecular biology, cell biology, protein & proteomics and microbiology. Reagent amounts scale to your number of samples, steps have built-in timers, and every protocol has critical points, troubleshooting and safety notes.
Immunology
Cell isolation, flow cytometry, T-cell assays, ELISA and EAE.
12 protocolsSingle-cell suspension from mouse spleen and lymph nodes
Mechanical dissociation of spleen and lymph nodes, red-cell lysis and counting — the starting point for flow cytometry, cell sorting and culture.
ImmunologyPBMC isolation by density-gradient centrifugation
Isolate peripheral blood mononuclear cells from whole blood using Ficoll-Paque or Lymphoprep (density 1.077 g/mL).
ImmunologySurface staining for flow cytometry
Viability dye, Fc block and a titrated antibody cocktail for surface markers, with the controls you need for clean gating.
ImmunologyIntracellular cytokine staining (PMA/ionomycin restimulation)
Restimulate T cells with PMA and ionomycin plus a protein-transport inhibitor, then fix, permeabilise and stain cytokines such as IL-17A, IFN-γ and IL-10.
ImmunologyTranscription factor staining (FoxP3, RORγt, T-bet)
Intranuclear staining with a FoxP3/transcription-factor buffer set after surface staining, compatible with cytokine staining.
ImmunologyCD4⁺ T-cell isolation by magnetic negative selection
Enrich untouched CD4⁺ (or naive CD4⁺) T cells from mouse spleen and lymph nodes with a biotin-antibody cocktail and streptavidin beads.
ImmunologyIn vitro T-helper polarisation (Th1, Th2, Th17, iTreg)
Activate naive CD4⁺ T cells with anti-CD3/CD28 under polarising cytokines and blocking antibodies to generate Th1, Th2, Th17 or induced Treg cells.
ImmunologyProliferation assay with CFSE or CellTrace dyes
Label cells with a dilution dye before culture and read the number of divisions by flow cytometry.
ImmunologySandwich ELISA for cytokines
Quantify a cytokine in supernatant, serum or plasma with capture and biotinylated detection antibodies, streptavidin-HRP and TMB.
ImmunologyActive EAE induction with MOG₃₅₋₅₅ in C57BL/6 mice
Induce experimental autoimmune encephalomyelitis with MOG₃₅₋₅₅ in complete Freund’s adjuvant plus pertussis toxin, and score clinical signs daily.
ImmunologyIsolation of CNS mononuclear cells with a Percoll gradient
Recover infiltrating immune cells and microglia from mouse brain and spinal cord after perfusion, enzymatic digestion and a 30%/70% Percoll gradient.
ImmunologyBone marrow-derived macrophages (BMDM)
Differentiate macrophages from mouse bone marrow with M-CSF (or L929-conditioned medium) over 6–7 days.
Molecular biology
DNA and RNA extraction, PCR, qPCR and cloning.
11 protocolsGenomic DNA from mouse tail or ear (HotSHOT)
A fast alkaline-lysis extraction that gives PCR-ready DNA from tail or ear biopsies in under an hour.
Molecular biologyPCR genotyping
Set up and run endpoint PCR to tell wild-type, heterozygous and homozygous (or floxed, Cre⁺) mice apart.
Molecular biologyAgarose gel electrophoresis
Cast, load and run an agarose gel to check PCR products, digests or plasmids, and image DNA bands.
Molecular biologyTotal RNA extraction with TRIzol
Phenol–guanidinium extraction of total RNA from cells or tissue, followed by isopropanol precipitation and quality checks.
Molecular biologycDNA synthesis (reverse transcription)
Convert equal amounts of total RNA into cDNA for qPCR, with gDNA removal and no-RT controls.
Molecular biologyqPCR with SYBR Green and the ΔΔCt method
Run SYBR Green qPCR in triplicate with melt curves, then calculate relative expression with ΔΔCt.
Molecular biologyPlasmid miniprep (alkaline lysis, spin column)
Purify plasmid DNA from a 1–5 mL overnight E. coli culture for sequencing, digestion or transfection.
Molecular biologyHeat-shock transformation of E. coli
Introduce plasmid DNA or a ligation into chemically competent E. coli and select transformants on antibiotic plates.
Molecular biologyRestriction digest and ligation cloning
Cut vector and insert with restriction enzymes, dephosphorylate the vector, purify and ligate at a set insert-to-vector molar ratio.
Molecular biologyGibson / HiFi DNA assembly
Join PCR fragments and a linearised vector with 20–40 bp overlaps in a single isothermal reaction.
Molecular biologyDNA and RNA quantification (NanoDrop and Qubit)
Measure nucleic-acid concentration and purity by UV absorbance, and use fluorescent dyes when accuracy matters.
Cell biology
Cell culture, transfection, imaging and viability assays.
10 protocolsThawing frozen cells
Rapidly thaw a cryovial, remove DMSO and plate cells for the best recovery.
Cell biologyPassaging adherent cells (trypsinisation)
Detach adherent cells with trypsin–EDTA, count and re-seed at the right split ratio.
Cell biologyCryopreservation of cells
Freeze cells in DMSO-containing medium at about −1 °C per minute and store them in liquid nitrogen.
Cell biologyCell counting and viability with trypan blue
Count live and dead cells on a hemocytometer and calculate concentration and viability.
Cell biologyMycoplasma testing by PCR
Screen culture supernatant for mycoplasma with a 16S rRNA PCR, with positive and negative controls.
Cell biologyPlasmid transfection with lipid reagents
Transfect adherent cells with plasmid DNA using a cationic lipid reagent (e.g. Lipofectamine-type) in a 24-well format.
Cell biologyImmunofluorescence staining of cultured cells
Fix, permeabilise, block and stain cells on coverslips with primary and fluorescent secondary antibodies, then counterstain nuclei.
Cell biologyFrozen tissue sections and immunofluorescence
Fix and cryoprotect tissue (for example mouse spinal cord or brain), embed in OCT, cut cryosections and stain them.
Cell biologyMTT cell viability assay
Measure metabolic activity as a proxy for viable cell number after treatment, in a 96-well plate.
Cell biologyApoptosis by Annexin V and propidium iodide
Distinguish live, early apoptotic and late apoptotic/necrotic cells by flow cytometry.
Protein & proteomics
Lysis, western blotting, Co-IP and mass-spectrometry sample prep.
10 protocolsProtein extraction with RIPA buffer
Lyse cultured cells or tissue in RIPA buffer with inhibitors and clear the lysate for western blotting.
Protein & proteomicsBCA protein assay (microplate)
Measure protein concentration against a BSA standard curve so you can load equal protein on gels.
Protein & proteomicsSDS-PAGE (polyacrylamide gel electrophoresis)
Denature, load and separate proteins by size on a Tris-glycine gel, with recipes for hand-cast gels.
Protein & proteomicsWestern blotting: transfer, immunodetection and ECL
Transfer proteins to PVDF or nitrocellulose, block, probe with antibodies and detect by chemiluminescence.
Protein & proteomicsStripping and reprobing a western blot
Remove bound antibodies with a mild glycine or a harsh SDS/β-mercaptoethanol buffer and probe the membrane again.
Protein & proteomicsCo-immunoprecipitation (Co-IP)
Pull down a protein with a specific antibody and protein A/G beads to detect its binding partners by western blot.
Protein & proteomicsCoomassie staining of protein gels
Stain SDS-PAGE gels with Coomassie R-250 (classic) or colloidal G-250 (more sensitive, mass-spectrometry compatible).
Protein & proteomicsIn-solution trypsin digestion for LC-MS/MS
Denature, reduce, alkylate and digest proteins with trypsin to make peptides for bottom-up proteomics.
Protein & proteomicsIn-gel trypsin digestion
Digest proteins from Coomassie-stained gel bands for protein identification by mass spectrometry.
Protein & proteomicsPeptide desalting with C18 StageTips
Clean up and concentrate digested peptides on C18 material before LC-MS/MS.
Microbiology
Media, plating, staining, growth and antibiotic susceptibility.
9 protocolsLB media and agar plates
Prepare and sterilise LB broth and pour selective agar plates with antibiotics.
MicrobiologyStreak plate for isolated colonies
Use the quadrant streak method to obtain single colonies from a culture or a glycerol stock.
MicrobiologyGram staining
Classify bacteria as Gram-positive or Gram-negative by crystal violet–iodine retention and safranin counterstain.
MicrobiologyBacterial growth curve by OD₆₀₀
Follow culture growth by optical density and calculate the doubling time in exponential phase.
MicrobiologyViable counts (CFU) by serial dilution and plating
Make 10-fold serial dilutions, plate them and count colonies to calculate colony-forming units per mL.
MicrobiologyBacterial glycerol stocks
Store bacterial strains long term at −80 °C in glycerol and revive them without thawing the stock.
MicrobiologyChemically competent E. coli (CaCl₂ method)
Make competent E. coli for routine heat-shock transformation from a mid-log culture.
MicrobiologyAntibiotic susceptibility by disk diffusion (Kirby–Bauer)
Test bacterial susceptibility by measuring inhibition zones around antibiotic disks on Mueller–Hinton agar.
MicrobiologyMinimum inhibitory concentration (broth microdilution)
Find the lowest antibiotic concentration that prevents visible bacterial growth, using two-fold dilutions in a 96-well plate.
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These are general starting-point protocols written for SolvesEverything. Always follow your institution’s approved protocols, safety rules and manufacturers’ instructions.