ESO wikiadmin at 14:00, 20 May 2025

2025-05-20T14:00:22Z

← Older revision		Revision as of 16:00, 20 May 2025
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	{\| class="wikitable"		{\| class="wikitable"
	\|+		\|+
	!		!Materials
	!
	!
	!		!
	\|-		\|-
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	FCS (heat-inactivated, final concentration: 5%)		FCS (heat-inactivated, final concentration: 5%)
	5% Natriumazid (final concentration: 0.1%)		5% Natriumazid (final concentration: 0.1%)
	\|
	\|
	\|-		\|-
	\|1st antibody:		\|1st antibody:
	\|primary antibody (approx. dilution 1:200)		\|primary antibody (approx. dilution 1:200)
	\|
	\|
	\|-		\|-
	\|2nd antibody:		\|2nd antibody:
	\|secondary antibody (approx. dilution 1:500)		\|secondary antibody (approx. dilution 1:500)
	fluorescence- labelled		fluorescence- labelled
	\|
	\|
	\|-		\|-
	\|controls:		\|controls:
Line 28:		Line 20:
	sample with secondary antibody only		sample with secondary antibody only
	if possible untransfected cells stained with both antibodies		if possible untransfected cells stained with both antibodies
	\|
	\|
	\|}		\|}

Line 35:		Line 25:
	Preparation of cells:		Preparation of cells:

	§ aspirate medium and wash twice with 5-10 mL 1xPBS		* aspirate medium and wash twice with 5-10 mL 1xPBS
			* detach cells with 1 mL Trypsin/EDTA
	§ detach cells with 1 mL Trypsin/EDTA		* inactivate Trypsin/EDTA with 3 mL fresh medium
			* transfer the cells into a 15 mL centrifuge tube and centrifuge 3 min at 600 rpm
	§ inactivate Trypsin/EDTA with 3 mL fresh medium		* discard supernatant and resuspend the cell pellet in FACS buffer
			* centrifuge again, resuspend cells in FACS buffer and determine cell count
	§ transfer the cells into a 15 mL centrifuge tube and centrifuge 3 min at 600 rpm		* distribute into Eppendorf tubes (106 cells/tube)

	§ discard supernatant and resuspend the cell pellet in FACS buffer

	§ centrifuge again, resuspend cells in FACS buffer and determine cell count

	§ distribute into Eppendorf tubes (106 cells/tube)


	~~Staining (perform all steps on ice):~~

	~~§ pellet the cells by centrifugation 2 min, 2500 rpm, 4°C~~

	~~§ resuspend pelled in 300 µL FACS buffer~~

	~~§ add 1st antibody at desired concentration~~

	~~§ incubate 1-1.5h on ice, centrifuge 2 min, 2500 rpm, 4°C~~

	~~§ discard supernatant and resuspend the pellet in 600 µL FACS buffer, centrifuge again as above~~

	~~§ wash 3 times with 300 µL FACS buffer, at the end resuspend in 300 µL FACS buffer~~

	~~§ add 2nd antibody at desired concentration~~

	~~§ incubate max. 30 min on ice (keep samples dark!)~~

	~~§ wash twice with 600 µL FACS buffer~~

	~~§ during centrifugation prepare FACS tubes with 600 µL FACS buffer~~

	~~§ resuspend pellet in 600 µL FACS buffer and transfer to prepared FACS tubes~~		=== Staining (perform all steps on ice): ===

	§ measure samples		* pellet the cells by centrifugation 2 min, 2500 rpm, 4°C
			* resuspend pelled in 300 µL FACS buffer
			* add 1st antibody at desired concentration
			* incubate 1-1.5h on ice, centrifuge 2 min, 2500 rpm, 4°C
			* discard supernatant and resuspend the pellet in 600 µL FACS buffer, centrifuge again as above
			* wash 3 times with 300 µL FACS buffer, at the end resuspend in 300 µL FACS buffer
			* add 2nd antibody at desired concentration
			* incubate max. 30 min on ice (keep samples dark!)
			* wash twice with 600 µL FACS buffer
			* during centrifugation prepare FACS tubes with 600 µL FACS buffer
			* resuspend pellet in 600 µL FACS buffer and transfer to prepared FACS tubes
			* measure samples

ESO wikiadmin: Created page with "{| class="wikitable" |+ ! ! ! ! |- |FACS buffer: |1xPBS FCS (heat-inactivated, final concentration: 5%) 5% Natriumazid (final concentration: 0.1%) | | |- |1st antibody: |primary antibody (approx. dilution 1:200) | | |- |2nd antibody: |secondary antibody (approx. dilution 1:500) fluorescence- labelled | | |- |controls: |sample with irrelevant primary antibody and secondary antibody sample with secondary antibody only if possible untransfected cells stained with both antib..."

2025-05-20T08:57:19Z

Created page with "{| class="wikitable" |+ ! ! ! ! |- |FACS buffer: |1xPBS FCS (heat-inactivated, final concentration: 5%) 5% Natriumazid (final concentration: 0.1%) | | |- |1st antibody: |primary antibody (approx. dilution 1:200) | | |- |2nd antibody: |secondary antibody (approx. dilution 1:500) fluorescence- labelled | | |- |controls: |sample with irrelevant primary antibody and secondary antibody sample with secondary antibody only if possible untransfected cells stained with both antib..."

New page

{| class="wikitable"
|+
!
!
!
!
|-
|FACS buffer:
|1xPBS
FCS (heat-inactivated, final concentration: 5%)
5% Natriumazid (final concentration: 0.1%)
|
|
|-
|1st antibody:
|primary antibody (approx. dilution 1:200)
|
|
|-
|2nd antibody:
|secondary antibody (approx. dilution 1:500)
fluorescence- labelled
|
|
|-
|controls:
|sample with irrelevant primary antibody and secondary antibody
sample with secondary antibody only
if possible untransfected cells stained with both antibodies
|
|
|}

=== Procedure ===
Preparation of cells:

§ aspirate medium and wash twice with 5-10 mL 1xPBS

§ detach cells with 1 mL Trypsin/EDTA

§ inactivate Trypsin/EDTA with 3 mL fresh medium

§ transfer the cells into a 15 mL centrifuge tube and centrifuge 3 min at 600 rpm

§ discard supernatant and resuspend the cell pellet in FACS buffer

§ centrifuge again, resuspend cells in FACS buffer and determine cell count

§ distribute into Eppendorf tubes (106 cells/tube)

Staining (perform all steps on ice):

§ pellet the cells by centrifugation 2 min, 2500 rpm, 4°C

§ resuspend pelled in 300 µL FACS buffer

§ add 1st antibody at desired concentration

§ incubate 1-1.5h on ice, centrifuge 2 min, 2500 rpm, 4°C

§ discard supernatant and resuspend the pellet in 600 µL FACS buffer, centrifuge again as above

§ wash 3 times with 300 µL FACS buffer, at the end resuspend in 300 µL FACS buffer

§ add 2nd antibody at desired concentration

§ incubate max. 30 min on ice (keep samples dark!)

§ wash twice with 600 µL FACS buffer

§ during centrifugation prepare FACS tubes with 600 µL FACS buffer

§ resuspend pellet in 600 µL FACS buffer and transfer to prepared FACS tubes

§ measure samples

Staining of cells for flow cytometry - Revision history

ESO wikiadmin at 14:00, 20 May 2025